Metabolic and Pharmaceutical Aspects of Fluorinated Compounds
- Benjamin M. Johnson*Benjamin M. Johnson*Phone: (617) 494-7349. Email: [email protected]Pharmaceutical Candidate Optimization, Bristol Myers Squibb Company, 100 Binney Street, Cambridge, Massachusetts 02142, United StatesMore by Benjamin M. Johnson,
- Yue-Zhong ShuYue-Zhong ShuPharmaceutical Candidate Optimization, Bristol Myers Squibb Company, Route 206 and Province Line Road, Princeton, New Jersey 08543, United StatesMore by Yue-Zhong Shu,
- Xiaoliang ZhuoXiaoliang ZhuoPharmaceutical Candidate Optimization, Bristol Myers Squibb Company, 100 Binney Street, Cambridge, Massachusetts 02142, United StatesMore by Xiaoliang Zhuo, and
- Nicholas A. MeanwellNicholas A. MeanwellDiscovery Chemistry Platforms, Small Molecule Drug Discovery, Bristol Myers Squibb Company, Route 206 and Province Line Road, Princeton, New Jersey 08543, United StatesMore by Nicholas A. Meanwell
Copyright © 2020 American Chemical Society
Abstract
The applications of fluorine in drug design continue to expand, facilitated by an improved understanding of its effects on physicochemical properties and the development of synthetic methodologies that are providing access to new fluorinated motifs. In turn, studies of fluorinated molecules are providing deeper insights into the effects of fluorine on metabolic pathways, distribution, and disposition. Despite the high strength of the C–F bond, the departure of fluoride from metabolic intermediates can be facile. This reactivity has been leveraged in the design of mechanism-based enzyme inhibitors and has influenced the metabolic fate of fluorinated compounds. In this Perspective, we summarize the literature associated with the metabolism of fluorinated molecules, focusing on examples where the presence of fluorine influences the metabolic profile. These studies have revealed potentially problematic outcomes with some fluorinated motifs and are enhancing our understanding of how fluorine should be deployed.
SPECIAL ISSUE
This article is part of the
Introduction
ARTICLE SECTIONS
The inclusion of fluorine in drugs, drug candidates, and agricultural chemicals continues to grow, a trend that is being driven by a deeper understanding of this remarkable element and its effects on molecular properties and biological activity.(1−14) The first fluorinated drug was introduced in 1955, and with the exceptions of 1960–1969 and 2000–2009, the presence of fluorine in approved drugs has increased every decade since, as summarized by the statistics presented in Figure 1. Many new synthetic methods to install fluorine have been developed over the last 15 years, providing access to a range of interesting and potentially useful fluorinated motifs.(15−20) The frequent use of fluorine in both drug and agricultural chemical design is also a function of its wide ranging effects on molecular properties.(1−5,9−12) Patterns of fluorination can enhance potency and/or specificity, increase membrane permeability, modulate metabolism, moderate the pKa of proximal functionalities, influence conformation, stabilize inherently reactive functionalities, and create higher-order bioisosteres that offer advantages, all of which are dependent on context.(1−8,21,22) In turn, studies of the metabolism and disposition of fluorinated compounds have advanced our knowledge and understanding of metabolic pathways. These efforts have not only provided insight into why fluorine can be advantageous but also identified structural arrangements that can present cryptic liabilities.(21,23) In this Perspective, we provide a survey of the metabolism of fluorinated compounds and capture observations that highlight potentially problematic structural arrangements.
Figure 1
Figure 1. Fluorine-containing drugs approved by the U.S. Food and Drug Administration through the end of 2019.
A common application of fluorinated motifs in drug design is to confer enhanced metabolic stability or influence the metabolism of a molecule. This application takes advantage of both the small size of the fluorine atom, which falls between that of a hydrogen atom and an oxygen atom, and the strength of the carbon–fluorine bond, which, at 98–115 kcal/mol, is the strongest of all of the common carbon–heteroatom bonds.(24) By comparison, C–H bonds (89–110 kcal/mol), C–Cl bonds (74–97 kcal/mol), and C–CH3 bonds (77–101 kcal/mol) are of lower strength.(25) However, while the C–F bond is strong and fluorine itself is a relatively poor leaving group (Table 1), there are many circumstances in which fluorine is not chemically inert.(26,27) This observation extends to alkyl, alkenyl, and aryl fluorides and encompasses mono-, di-, and trifluorinated substituents, which can be intrinsically chemically reactive or activated during metabolism. As an example, (S)-γ-fluoroleucine (1) readily cyclizes to the lactone 2 with the expulsion of fluoride (Scheme 1), which is remarkable since the fluorine is bound to a quaternary center and the carboxylate moiety is a relatively poor nucleophile. This reaction leads to significant chemical degradation of 1 in vivo, which limits the potential of the [18F] isotope to be used as a tracer for positron emission tomography (PET) studies of tumors.(28) As another example of this phenomenon, (S)-γ-fluoroleucine derivative 3, 
a synthetic precursor of the cathepsin K inhibitor odanacatib (4), must be stored at 4 °C to prevent the occurrence of a similar cyclization.(29,30) Fluorinated motifs that are converted enzymatically to intermediates that expel fluoride have been exploited in the design of mechanism-based inhibitors (MBIs) of enzymes, while the metabolism of a fluorinated molecule can set the stage for the elimination of fluoride, leading to the generation of electrophilic products that may react with biological nucleophiles.(31) Moreover, the metabolism of fluorinated compounds can be extensive, with processing through multiple consecutive steps that can culminate in the release of low-molecular-weight and structurally simple fluorinated toxins.
a synthetic precursor of the cathepsin K inhibitor odanacatib (4), must be stored at 4 °C to prevent the occurrence of a similar cyclization.(29,30) Fluorinated motifs that are converted enzymatically to intermediates that expel fluoride have been exploited in the design of mechanism-based inhibitors (MBIs) of enzymes, while the metabolism of a fluorinated molecule can set the stage for the elimination of fluoride, leading to the generation of electrophilic products that may react with biological nucleophiles.(31) Moreover, the metabolism of fluorinated compounds can be extensive, with processing through multiple consecutive steps that can culminate in the release of low-molecular-weight and structurally simple fluorinated toxins.Table 1. Relative Rates of Reaction of 1-Halo-3-methylbutanes with NaOMe in MeOH to Afford 1-Methoxy-3-methylbutane(7)
| X | relative rate of reaction |
|---|---|
| F | 1 |
| Cl | 71 |
| Br | 3500 |
| I | 4500 |
Scheme 1
Scheme 1. (S)-γ-Fluoroleucine (1) Converts Spontaneously to Amino Lactone 2 with Loss of HF
1. Fluoroacetic Acid and Other Small Fluorinated Compounds as Toxins and Metabolic Byproducts
ARTICLE SECTIONS
1.1. Fluoroacetic Acid Biosynthesis
Fluoroacetic acid (5) is an elementary fluorinated compound that is profoundly toxic in vivo and can be produced by several metabolic pathways from precursors 6–12′ (Figure 2). Accordingly, there are documented examples in which the release of 5 or a precursor during the metabolism of a fluorinated drug candidate has presented toxicological problems. First synthesized in 1896, 5 was subsequently discovered to occur naturally in plants and bacteria.(32,33) The biosynthetic pathway leading to 5 is captured in Scheme 2, with the fluorinase-dependent conversion of S-adenosylmethionine (13) to 5′-fluoro-5′-deoxyadenosine (14) representing the key step in forging the carbon–fluorine bond.(34) Theoretical studies suggest that the fluorinase enzyme reduces the barrier to C–F bond formation by 9.3–11.7 kcal/mol and chaperones the poorly nucleophilic, basic fluoride toward attack onto C-5′ of the ribose of S-adenosylmethionine.(35) This process is favored kinetically over attack onto the other two possible carbon atoms, whereas deprotonation to afford a sulfur ylide is calculated to be thermodynamically unfavorable.(36−38) The binding of fluoride to the enzyme is believed to precede the binding of S-adenosylmethionine. The reaction has been modeled to proceed by an SN2 process that entails a formal inversion of configuration at C-5′, although the proposed trajectory has fluoride approaching at a slightly suboptimal 164° angle of attack.(37) Hydrogen-bonding interactions between fluoride and the backbone N–H and side-chain O–H groups of Ser158 and Thr80 compensate for the penalty associated with desolvating fluoride from its aqueous environment.(37) The ribose moiety is held in place by additional hydrogen-bonding interactions between the 2′,3′-diol moiety and Asp16, the side chain N–H of Trp50, and the backbone N–H of Tyr77, all of which help to constrain the furan ring.(36−38) The release of 5 from 14 follows a pathway that is initiated by exchange of the adenine base for phosphate, a process catalyzed by purine nucleoside phosphorylase, to provide 5-fluoro-5-deoxy-d-ribose-1-phosphate (15). Phosphate 15 is converted to acyclic 16 by an isomerase, and an aldolase converts 16 to 17 via a retro-aldol reaction. This step results in the concomitant release of fluoroacetaldehyde (6), which is oxidized by aldehyde dehydrogenase (ADH) to complete the synthesis of 5. A pyridoxal 5′-phosphate (PLP)-dependent transaldolase uses l-threonine as a substrate to exchange 6 for a molecule of acetaldehyde, producing 4-fluorothreonine (18) as the only other known naturally occurring fluorinated compound.(39) Additionally, fluorinated uracils have been isolated from marine sponges, but these compounds may have accumulated due to exposure to an industrial effluent.(36,40)
Figure 2
Figure 2. Fluoroacetic acid (5) and known metabolic precursors 6–12.
Scheme 2
Scheme 2. Biosynthesis of 5 and 18 and (Inset) Details of the Transition State as Proposed by Theoretical, Structural, and Kinetic Studies
1.2. Fluoroacetic Acid Toxicity
Fluoroacetic acid (5) is toxic to mammals, with the lethal dose in humans ranging from 2 to 10 mg/kg. Rats (0.1–5 mg/kg) and dogs (0.05 mg/kg) are incrementally more sensitive.(41) As a consequence, the sodium salt of 5 (also known as “Compound 1080”) is used as a rodenticide.(42) The toxicity associated with 5 is fully recapitulated by the aldehyde 6, the methyl (7), propyl (8), and isopropyl (9) esters, the primary amide 10, and 2-fluoroethanol (11) (Figure 2). These observations are consistent with the notion that these molecules act as prodrugs of 5 in vivo.(43) 2-Fluoroethyl 2-fluoroacetate (12) is twice as toxic as 5, reflecting the dual source of the toxin from this ester.
The toxicity of 5 is caused by its entry into the Krebs cycle and conversion to a potent inhibitor of the enzyme aconitase.(41,44) The normal function of aconitase is to convert citric acid (19) to isocitric acid (21) by the pathway outlined in Scheme 3A. The initial step is the dehydration of 19 to afford cis-aconitate (20), which is believed to flip in the active site of the enzyme to set the stage for the addition of H2O to deliver 21.(44) However, aconitase also catalyzes the dehydration of (−)-erythro-(2R,3R)-2-fluorocitric acid (24) in a manner that is analogous to its dehydration of the natural substrate (Scheme 3B). Remarkably, the Claisen condensation catalyzed by citrate synthase between 22, the CoA ester of 5, and 2-oxosuccinic acid (23) produces only 24 and none of the other three possible diastereomers. Hydration of the olefin of 25 by aconitase is usurped when the addition of H2O precipitates an SN2′-like process that ejects the allylic fluoride, generating 4-hydroxy-trans-aconitate (26), a potent but reversible and tight-binding inhibitor of aconitase (Scheme 3B).(41,44,45) Turnover of 24 by aconitase produces a single molecule of fluoride per enzyme, indicative of efficient enzymatic inactivation.
Scheme 3
Scheme 3. Biochemical Reactions of Native and Fluorinated Substrates Catalyzed by Aconitasea
a(A) Dehydration of citric acid (19) to form 20 is followed by rehydration to give isocitric acid (21). (B) Compound 5 is converted to fluoroacetyl-CoA (22), which reacts by a Claisen condensation with 23 to give (−)-erythro-(2R,3R)-2-fluorocitric acid (24) in a stereospecific manner. Dehydration of 24 to give 25 is followed by a flip in the active site as a prelude to the addition of H2O. This addition produces 4-hydroxy-trans-aconitate (26), a potent and tight-binding inhibitor of aconitase that represents the ultimate toxicant produced by the biochemical transformation of 5.
Interestingly, aconitase also accepts (+)-erythro-(2R,3R)-2-fluorocitric acid (27) as a substrate but continues to produce fluoride so long as this substrate remains available.(41,44) The explanation for this finding invokes the binding of 27 to aconitase with an orientation that is the reverse of 24, thereby producing 28 (Scheme 4). A flip of 28 in the active site followed by hydration affords the fluorohydrin 29, which eliminates HF and degrades to oxalosuccinic acid (30). Decarboxylation of 30 produces α-ketoglutaric acid (31), completing a biochemical sequence in which no strong inhibitors of aconitase are formed.
Scheme 4
Scheme 4. Metabolism of (+)-erythro-(2S,3S)-2-Fluorocitric Acid (27) by Aconitasea
a(+)-erythro-(2S,3S)-2-Fluorocitric acid (27) is subject to elimination of H2O to afford 28, which flips in the enzyme active site to set the stage for the addition of H2O to produce 29. This compound collapses with loss of HF to give oxalosuccinic acid (30), which decarboxylates to afford α-ketoglutaric acid (31).
Many of the features of poisoning by 5 include direct and indirect consequences of impaired oxidative metabolism. Energy production is reduced, and intermediates of the Krebs cycle subsequent to 19 are depleted.(41,46) Concomitantly, the inactivation of aconitase leads to a rapid elevation of the levels of 19 in blood. The symptoms of poisoning associated with 5, including nausea, vomiting, abdominal pain, sweating, apprehension, confusion, and agitation, are exacerbated by the accumulation of 19 in tissues and typically occur within an hour of ingestion. These symptoms are accompanied by multiple neurological, metabolic, and respiratory disturbances that are eventually fatal.(41,47) (−)-erythro-(2R,3R)-2-Fluorocitric acid (24) is also an irreversible inhibitor of the mitochondrial citric acid transport protein by a mechanism that involves covalent modification of two protein fractions that are associated with the mitoplast of liver, kidney, brain, and heart tissue.(48) This covalent reaction does not involve the loss of fluoride and is catalyzed by an enzyme in a Mn2+-dependent fashion. The protein can be released by treatment with hydroxylamine in a reaction process that produces the hydroxamic acid derivative of 24.(48)
1.3. Metabolism and Release of Fluoroacetic Acid
1,3-Difluoro-2-propanol (32) is also used as a rodenticide. The symptoms of poisoning mimic those of 5, although there is a lag time for the onset attributed to the requirement for metabolism through several steps to release the toxin. Initially 32 is converted to 1,3-difluoroacetone (33) by alcohol dehydrogenase (ALDH), a metabolic step that can be inhibited by 4-methylpyrazole. Both 32 and 33 are metabolized to 24 in rats, although the metabolic pathway that has been proposed for this reaction is mechanistically unsatisfactory since it fails to account for the extrusion of a carbon atom of 33 in a way that is chemically feasible.(49)Scheme 5 suggests an alternative mechanism that invokes a Baeyer–Villiger-type process to generate fluoromethyl 2-fluoroacetic acid (12′) as an intermediate, an apparently unknown compound that can function as a precursor to 5 and then 22.(49−55) While the enzyme that catalyzes this reaction is also unknown, both flavin-containing monooxygenase 5 (FMO5) and the hydroperoxy form of P450 enzymes are known to catalyze similar oxidative rearrangements.(56−59) Hydrolytic cleavage of 12′ leads presumably to the release of formaldehyde and fluoride.
Scheme 5
Scheme 5. Potential Mechanism for the Metabolic Activation of 1,3-Difluoro-2-propanol (32) to Form Fluoroacetic Acid CoA Estera
aThis process is hypothesized to proceed via oxidation of 32 by ALDH to give 1,3-difluoroacetone (33) followed by a Baeyer–Villiger-type process to generate ester 12′. This ester would be anticipated to convert readily to 5 by hydrolysis or otherwise undergo esterase-mediated cleavage to release 5, formaldehyde, and fluoride.
There have been several incidents during drug discovery campaigns where the metabolism of a compound resulted in the formation of 5 and produced toxicity and mortality in preclinical species.(60,61) For example, in a series of kinesin spindle protein (KSP) inhibitors explored for their potential to treat taxane-refractory solid tumors, moderating the basicity of the piperidine ring to a target pKa range of 6.5 to 8.0 was an important parameter to reduce efflux by P-glycoprotein (P-gp) in tumor cells (Figure 3).(60) Addressing P-gp efflux was a focus of this project, and this was assessed by screening compounds in cell lines that either overexpressed or did not express P-gp and defining the resulting ratio of EC50 values as the multidrug resistance protein 1 (MDR) ratio. The calculated MDR ratio for the methylated piperidine 34 was 21.2, well beyond the targeted ratio of 10 (Figure 3). The N-2-fluoroethyl derivative 35 provided the targeted control of piperidine basicity, whereas the less basic difluoroethyl homologue 36 did not. However, 35 was acutely toxic to rats following oral administration at a dose of 12 mg/kg. The identification of the N-dealkylated piperidine 37 as the major metabolite in rat liver microsomes (RLM) and hepatocytes led to the suggestion that the release of 6 would provide a metabolic precursor to 5, believed to be the ultimate toxicant (Scheme 6).(60) This problem was circumvented by installation of the fluorine atom within the piperidine ring, where the effect on the pKa of the heterocyclic N atom was dependent on whether the fluorine was oriented axially or equatorially. The axial isomer 39 (MK-0731) was 1.2 log10 units less basic than the equatorial isomer 38, with the former offering the preferred profile and being selected for a phase I clinical trial.(60)
Figure 3
Figure 3. Example of the tactical deployment of fluorine to modulate the basicity in piperidine-based KSP inhibitors.
Scheme 6
Scheme 6. Major Metabolic Pathway for 35 in RLM and Hepatocytes
The peptide-based fluoromethyl ketone Z-Phe-Ala-CH2F (40) offers another example of the metabolic release of 5.(61) This compound was designed as an MBI of cysteine proteases and examined as a potential treatment for osteoarthritis. Both oral and intravenous administration of 40 to rodents produced 5 in the heart, liver, and kidney.(61) Notably, following oral administration of 40 at 100 mg/kg, the concentrations of 5 in these tissues reached toxic levels of 0.2 to 0.9 μg/g of tissue. While a mechanistic explanation for the release of 5 from 40 was not offered, a Baeyer–Villiger rearrangement catalyzed by an FMO or a P450 enzyme may be responsible, as suggested by the mechanism presented in Scheme 7.(54,56) The initial addition reaction is facilitated by the electron-withdrawing fluorine substituent and involves the addition of a peroxy moiety, which is a known intermediate in the P450 catalytic cycle.(62) This moiety attacks the electrophilic carbonyl to afford an intermediate that rearranges to ester 41. Hydrolysis of 41 by esterases or peptidases in tissues would release 5 and the unstable aminal 42, which would collapse to acetaldehyde (43) and the amide Cbz-Phe-NH2 (44). The teaching in this example is that mono-α-fluoromethyl ketone derivatives should be deployed with caution, with additional steps taken to ensure that 5 is not liberated as the result of metabolism. In particular, it may be inadequate to address this concern by performing broad metabolite profiling studies using liquid chromatography and electrospray mass spectrometry (LC–MS), since 5 has a low molecular weight (78 Da) and could be easily overlooked using these methods. Hence, drug candidates containing this motif may require a targeted approach to measure the extent of 5 released in vivo. Such an approach would be warranted, for example, in the case of the autophagin-1 (ATG4B) inhibitor 45, which is believed to alkylate the catalytic cysteine (Cys74) of the enzyme to afford 46 by the mechanism illustrated in Scheme 8.(63−65)
Scheme 7
Scheme 7. Enzymatic Baeyer–Villiger Oxidation of Z-Phe-Ala-CH2F (40)
Scheme 8
Scheme 8. Covalent Inhibition of ATG4B by the Fluorinated Peptide Mimetic 45 to Afford Alkylated Enzyme 46
An [18F]-substituted PET tracer designed to image the cannabinoid receptor subtype 2 (CB2) in the central nervous system (CNS) was prepared from a series of carbazole-based ligands, with 47
demonstrating high affinity for the CB2 receptor and good selectivity over the CB1 subtype.(66) However, this tracer was metabolized rapidly in mice, with oxidative N-dealkylation postulated as one of the main metabolic pathways, raising a safety concern over the potential liberation of 6 and its subsequent oxidation to 5.(66) On the basis of these observations, a series of analogues with expanded fluoroalkyl moieties were synthesized and evaluated. The additional CH3 substituent in the side chain of 48 shielded the carbazole N atom from metabolism by P450 while retaining the high CB2/CB1 selectivity associated with 47. The extent of oxidative N-dealkylation of 48 in liver microsomes was reduced by two-thirds compared with 47, translating to improved exposure in vivo.(66) However, the N-dealkylation of 48 would release fluoroacetone, a compound that produces toxicity in vivo similar to that of 5. Presumably, this effect is a function of metabolism to methyl 2-fluoroacetate (7) via a Baeyer–Villiger-type process, as proposed for 1,3-difluoropropan-2-one (33) (Scheme 5).(67,68)
demonstrating high affinity for the CB2 receptor and good selectivity over the CB1 subtype.(66) However, this tracer was metabolized rapidly in mice, with oxidative N-dealkylation postulated as one of the main metabolic pathways, raising a safety concern over the potential liberation of 6 and its subsequent oxidation to 5.(66) On the basis of these observations, a series of analogues with expanded fluoroalkyl moieties were synthesized and evaluated. The additional CH3 substituent in the side chain of 48 shielded the carbazole N atom from metabolism by P450 while retaining the high CB2/CB1 selectivity associated with 47. The extent of oxidative N-dealkylation of 48 in liver microsomes was reduced by two-thirds compared with 47, translating to improved exposure in vivo.(66) However, the N-dealkylation of 48 would release fluoroacetone, a compound that produces toxicity in vivo similar to that of 5. Presumably, this effect is a function of metabolism to methyl 2-fluoroacetate (7) via a Baeyer–Villiger-type process, as proposed for 1,3-difluoropropan-2-one (33) (Scheme 5).(67,68)The anticancer drug 5-fluorouracil (49) was metabolized to 5 in rats following intraperitoneal (ip) administration (180 mg/kg) and in isolated perfused rat livers at exposures of 15 and 45 mg/kg (Scheme 9).(69) Specifically, 19F NMR analysis revealed the presence of 5 in the urine of rats (0.003% of the dose) and in the liver perfusate (0.03 to 0.1% of substrate concentration), in addition to other metabolites of 49. Perfusion of rat liver with α-fluoro-β-alanine (52), a known metabolite of 49, also produced 5 in the perfusate (0.07% of the substrate concentration), a finding that helped to confirm the complex catabolic pathway from 49 to 5 shown in Scheme 9. These results were recapitulated in patients treated with 49, whose urine contained 5 among other fluorinated metabolites.(70) Capecitabine (Xeloda, 59), an oral prodrug of 49 that demonstrates enhanced tumor selectivity, follows an activation pathway that involves unmasking by way of the metabolites 60 and 61 to form 49 preferentially in tumor tissues (Scheme 10).(71) By means of 19F NMR spectroscopy, low amounts of 5 were detected in the urine samples of patients treated with 59.(72) In human urine, the combination of 51 and 52 represented 50% of the administered dose, which is typically 150 or 500 mg. These observations have implicated the release of 5 in the cardiotoxicity and neurotoxicity associated with the clinical use of 49. The pathway summarized in Scheme 9 depends on the reduction of 49 by dihydropyrimidine dehydrogenase to give 50, which is subject to hydrolytic decomposition to afford first the urea 51 and then the amine 52. Oxidative deamination of 52 provides 53, which may either decarboxylate to give intermediate 6 or undergo oxidation to give 54. Both of these intermediates can then be converted directly to 5. It is instructive to note that on the basis of the metabolic pathway outlined in Scheme 9, the carbon atom of 49 that is incorporated into 6 may be derived not only from the C-4 carbonyl moiety but also prospectively from C-6.
Scheme 9
Scheme 9. Biotransformation of 49a
a(a) 5-Fluorouracil (49) is a substrate of both orotate phosphoribosyl transferase, which catalyzes the addition of a phosphorylated ribose moiety to yield 55, and dihydropyrimidine dehydrogenase, which carries out the initial step in the catabolic pathway that mediates the disposition of the drug. (b) The triphosphate metabolite 56 may be incorporated into DNA, leading to downstream single-strand breaks. (c) The monophosphate 57 reacts with thymidylate synthase and 5,10-methylene tetrahydrofolate, forming the covalent inactivated enzyme complex 58. (d) The multistep catabolic sequence proceeds through a postulated carbon–carbon bond cleavage and decarboxylation, culminating in the formation of 5, in which the acid group may be derived from either C-4 or C-6 of the starting material.
Scheme 10
Scheme 10. Metabolism of Capecitabine (59) to 49
Alternatively, 49 may enter the nucleotide biosynthesis pathway, generating a host of intermediates, including 55 en route to 56. Triphosphate 56 can either be incorporated into DNA or successively dephosphorylated to afford the monophosphate 57, which is a covalent inactivator of thymidylate synthase.(73) Inhibition of this enzyme involves nucleophilic attack by the catalytic cysteine residue onto C-6 of 57, mimicking the Michael addition process that occurs with the natural substrate. However, the presence of the fluorine atom usurps the normal process of methylation and leads to the stable covalent complex 58. The incorporation of 56 into DNA and the covalent modification of thymidylate synthase are two of the mechanisms by which 49 exerts its pharmacological activity.
Another example of a metabolic pathway leading to the inadvertent production of fluoroacetic acid was documented during the development of the hepatitis C virus (HCV) NS4B inhibitors 62–65 illustrated in Figure 4.(74,75) Safety evaluation of the lead HCV inhibitors 62 and 63 in rats revealed two problems associated with metabolic modification: N-dealkylation of the sulfonamide with presumed release of 1,3-difluoroacetone (33), a metabolic precursor to 5 (Scheme 5), and oxidative metabolism of the indole ring leading to several glutathione (GSH) conjugates that accounted for approximately 20% of the administered dose. In the design of the subsequent compound 64, installation of a fluorine substituent at C-5 of the indole attenuated metabolism of the heterocycle, while modification of the sulfonamide moiety addressed the release of 33. In the rat, 64 was metabolized primarily by N-dealkylation of the primary sulfonamide, releasing trifluoromethyl acetone, which was detected in plasma and bile. Additionally, a single GSH conjugate that amounted to <2.5% of the dose administered was formed, reflecting the influence of the indole 5-fluoro substituent. While this compound represented an improvement over 62, it was characterized by a long half-life in both the dog (149 h) and monkey (22 h), necessitating additional modification that led to the identification of 65. This compound exhibited more reasonable half-lives in the rat, dog, and monkey (7.5 to 8.9 h) and was selected for clinical evaluation.(75) The major metabolites derived from 65 in vivo were the result of benzylic oxidation and N-dealkylation of the sulfonamide.
Figure 4
Figure 4. Optimization of HCV NS4B inhibitors to avoid the production of 33, a metabolic precursor to 5.
1.4. Other Simple Fluorinated Compounds and Their Homologues
To understand how fluorine substitution alters the toxicology of small fluorinated aldehydes and acids, the effects of non-halogenated compounds are worth considering as a basis for comparison. Humans are exposed to acetaldehyde by the inhalation of tobacco smoke and emissions from industrial sources as well as from the consumption of ethanol. Acute and subacute inhalation of acetaldehyde in rats caused dose-dependent degenerative hyperplastic and metaplastic changes in the epithelium of the upper respiratory tract, accompanied by growth retardation and hematological changes.(76) In a chronic inhalation study, rats developed squamous cell carcinomas in the respiratory epithelium and adenocarcinomas in the olfactory epithelium.(77,78) Chronic consumption of alcohol led to the excessive hepatic disposition of acetaldehyde, an oxidative metabolite of ethanol and a culprit in the development of alcoholic liver diseases and hepatocellular carcinoma.(79) To some extent, acetaldehyde may be detoxified via conjugation with cysteinylglycine in vivo.(80) Acetaldehyde initiates mutagenic events by direct interactions with DNA and promotes tumor progression through various mechanisms, including elevated oxidative stress, damage to DNA repair mechanisms, and the formation of protein adducts that may elicit an immune response. On the other hand, acetic acid is a common chemical intermediate in the manufacture of organic compounds, insecticides, and food additives. It causes acute corrosion of the mucous membranes of the respiratory and gastrointestinal tracts through inhalation and ingestion, respectively, and dermal irritation through local absorption.(81) Acetic acid was not genotoxic in the Ames test or in the SOS chromotest,(82) and while it caused concentration-dependent chromosomal aberrations in Chinese hamster ovary K1 cells, this effect was ascribed to the reduced pH of the culture medium.(83) Interestingly, acetic acid also expedited cancer progression in a papilloma-bearing mouse model.(84)
While fluoroacetic acid (5) is toxic because of its facile entry into the Krebs cycle, the higher homologues difluoroacetic acid (66) and trifluoroacetic acid (67) 
presented different toxicological profiles in vivo.(85−89) Analogous to the production of 5 in vivo, 66 and 67 are potential byproducts that may be formed during oxidative or hydrolytic metabolism of small-molecule drug candidates. However, there are fewer reports of toxicity associated with these carboxylic acids. Developmental toxicological studies on the haloacetic acids 66, 67, and 68 using 3-6 somite staged CD-1 mouse embryo cultures revealed dysmorphogenesis after 24 h of exposure.(85,86) The observed effects on neural tube development ranged from prosencephalic hypoplasia to nonclosure defects throughout the cranial region. Exposure to 66–68 also affected optic development, produced malpositioned and/or hypoplastic pharyngeal arches, and perturbed heart development. In order to determine the relative toxicities of these agents, benchmark concentrations were calculated as the lower 95% confidence interval of the concentration that produced a 5% increase in neural tube defects. The benchmark concentrations occurred over a wide range, with 66 the least toxic (5912 μM), 67 (3652 μM) and 5 (692 μM) incrementally more problematic, and 68 (2.7 μM) the most toxic. Interestingly, the chlorinated acids 69 (2452 μM) and 70 (1336 μM) disrupted development in vivo but were among the least toxic in vitro.
presented different toxicological profiles in vivo.(85−89) Analogous to the production of 5 in vivo, 66 and 67 are potential byproducts that may be formed during oxidative or hydrolytic metabolism of small-molecule drug candidates. However, there are fewer reports of toxicity associated with these carboxylic acids. Developmental toxicological studies on the haloacetic acids 66, 67, and 68 using 3-6 somite staged CD-1 mouse embryo cultures revealed dysmorphogenesis after 24 h of exposure.(85,86) The observed effects on neural tube development ranged from prosencephalic hypoplasia to nonclosure defects throughout the cranial region. Exposure to 66–68 also affected optic development, produced malpositioned and/or hypoplastic pharyngeal arches, and perturbed heart development. In order to determine the relative toxicities of these agents, benchmark concentrations were calculated as the lower 95% confidence interval of the concentration that produced a 5% increase in neural tube defects. The benchmark concentrations occurred over a wide range, with 66 the least toxic (5912 μM), 67 (3652 μM) and 5 (692 μM) incrementally more problematic, and 68 (2.7 μM) the most toxic. Interestingly, the chlorinated acids 69 (2452 μM) and 70 (1336 μM) disrupted development in vivo but were among the least toxic in vitro.The production of trifluoroacetaldehyde (71) in vivo as a metabolic intermediate was associated with testicular toxicity and was thought to be responsible for the lesion produced following the administration of trifluoroethanol (72).(87,88) In the rat, the damage to spermatogonia and spermatocytes following a single dose of 72 (25 mg/kg ip) was apparent within 8 h. The lesion was first observed during the late pachytene stage of spermatogenesis. Interestingly, 71 and 72 were more potent toxins in vivo than 67, which produced the same lesion but only at higher doses.(87) The metabolism and toxicity of 72 in rats was described previously.(88) Deuterated 72 was less toxic (LD50 = 0.44 g/kg) than 72 (LD50 = 0.21 g/kg), reflecting its 4.2-fold lower intrinsic clearance (Vmax/Km) in liver microsomes, a process that involves the oxidation of the alcohol to the aldehyde. In contrast, deuteration of 71 did not affect either its lethality or rate of metabolism.
N-Trifluoroethyl moieties can be N-dealkylated by P450 enzymes in vivo to release 71, as demonstrated by studies with piperidine derivatives based on 73 (Scheme 11; it should be noted that the full structures of the substrates were not disclosed).(89) Following administration of 73 to rats, testicular toxicosis was apparent. The toxicological effects correlated with exposure to 71 and targeted both the epididymis, which became filled with cellular debris, and the spermatocytes, which experienced necrosis.(89) A similar safety study performed with the N-tetrazolyl analogue 75 revealed no evidence of testicular injury, providing further support for the role of 71 in the development of the testicular lesion.(89) This observation heightens concern for the formation of 71 during the metabolism of O- and S-trifluoroethylated compounds.
Scheme 11
Scheme 11. Compounds Containing N-Trifluoroethyl Groups May Be Metabolized In Vivo to Release 71, Which Causes Testicular Toxicity
Finally, fluoride itself is known to carry both benefits and risks.(90,91) On the one hand, fluoride is used widely in dental health for the prevention of tooth decay (dental caries or cavities). Fluoridated water (approximately 0.5 to 0.7 mg/L fluoride) and fluoride-containing toothpaste (approximately 1000 to 2000 ppm) and mouth rinses (approximately 0.05% or 225 ppm) are all effective fluoride supplements that are used for this purpose. On the other hand, the distribution of fluoride into tissue is thought to be nonsaturable, and long-term exposure to abundant sources of fluoride can lead to health problems. Many of the consequences of excessive fluoride exposure are also irreversible. Since fluoride distributes into bones, dental fluorosis provides an early indication of chronic fluoride toxicity. This condition is characterized by hypomineralization, increased porosity of surface enamel, and tooth discoloration and damage. Severe chronic fluorosis is also manifested in the skeleton, where increased bone density and abnormal bone outgrowths are observed. Clinical symptoms of skeletal fluorosis may include joint stiffness and pain, deformities in the joints and spine, and neurological problems. Crippling skeletal fluorosis is rare and associated with intake of highly fluoridated water (greater than 10 mg/L). Such effects can also arise in connection with metabolic release of fluoride from drugs that are administered at high doses (vide infra).
1.5. Defluorinating Enzymes
Halogenases with the capacity to defluorinate 5 have been characterized in bacteria, plants, and several mammalian species.(92−95) The mammalian and bacterial enzymes employ an Asp-His-Asp catalytic triad in which one of the Asp residues acts as a nucleophile to displace the fluoride by an SN2-type mechanism. Two proximal arginine residues stabilize the negative charge on the carboxylic acid of 5. The departure of fluoride is facilitated by the side chains of proximal Tyr, Trp, and His residues that are believed to lower the activation barrier for fluoride release via hydrogen-bonding interactions (Scheme 12). The subsequent hydrolysis of the aspartate ester releases glycolic acid and restores the enzyme to its native state.(93−99) The architecture of this halide pocket confers specificity for fluoride over chloride, such that chloroacetic acid is processed more slowly.
Scheme 12
Scheme 12. Mechanism of Defluorination of 5 by Mammalian and Bacterial Defluorinase Enzymes
Some mammalian defluorinase activity has been found to depend on GSH, leading to the suggestion that this enzyme may be a glutathione S-transferase (GST).(92,100) Available evidence indicates that GST zeta (GSTZ) is involved in catalyzing the dehalogenation of some α-haloacids.(100) However, the rate of conversion of 5 to S-(carboxymethyl)glutathione (76) 
by GSTZ was relatively low (172 nmol min–1 (mg of protein)−1). Higher rates of catalysis were measured with other substrates, including racemic 2-chloropropionic acid (1655 nmol min–1 (mg of protein)−1), 2-bromopropionic acid (2142 nmol min–1 (mg of protein)−1), and 2-iodopropionic acid (2532 nmol min–1 (mg of protein)−1). Similar findings were obtained in another study that found GSTZ1C to be the only GST isoform to possess catalytic activity to release fluoride from 5, although the rate of catalysis (210 nmol min–1 (mg of protein)−1) was lower than for other model substrates, including dichloroacetic acid (69) and chlorofluoroacetic acid (77).(92) The defluorination activity resided mainly in the cytosolic fraction of rat liver and was inhibited noncompetitively by 1-chloro-2,4-dinitrobenzene. Interestingly, this study found GSTZ1 to account for only 3% of all of the defluorination activity toward 5 in rat cytosol; thus, GSTZ1 appears to contribute marginally to the overall detoxification of 5.(92)
by GSTZ was relatively low (172 nmol min–1 (mg of protein)−1). Higher rates of catalysis were measured with other substrates, including racemic 2-chloropropionic acid (1655 nmol min–1 (mg of protein)−1), 2-bromopropionic acid (2142 nmol min–1 (mg of protein)−1), and 2-iodopropionic acid (2532 nmol min–1 (mg of protein)−1). Similar findings were obtained in another study that found GSTZ1C to be the only GST isoform to possess catalytic activity to release fluoride from 5, although the rate of catalysis (210 nmol min–1 (mg of protein)−1) was lower than for other model substrates, including dichloroacetic acid (69) and chlorofluoroacetic acid (77).(92) The defluorination activity resided mainly in the cytosolic fraction of rat liver and was inhibited noncompetitively by 1-chloro-2,4-dinitrobenzene. Interestingly, this study found GSTZ1 to account for only 3% of all of the defluorination activity toward 5 in rat cytosol; thus, GSTZ1 appears to contribute marginally to the overall detoxification of 5.(92)While the role of mammalian GSTs in the defluorination of 5 has been suggested, there is evidence for the involvement of other enzymes as well. One study indicated that the defluorinase activity present in mouse liver cytosol did not bind to a GSH affinity column, a tool used widely for the separation of GST isozymes.(101) Protein chromatofocusing and immunoprecipitation studies also indicated that the defluorinase activity was clearly distinct from GST isozymes. This enzyme preparation catalyzed the defluorination of fluoroacetamide 10 as well as acid 5, although the rate of the former reaction was approximately 10-fold lower. A similar difference in the rate of defluorination between acid 5 and amide 10 was observed when crude rat and mouse liver supernatants were used.(102)
The fate of 5 in rodents was investigated alongside a series of analogues of 5, which were administered ip at doses ranging from 2 to 5 mg/kg together with appropriate antidotes.(103)19F NMR spectra of urine and kidney samples collected from these animals revealed the presence of fluoride. Following administration of isotopically labeled derivatives of 5, a number of conjugates related to 76 and their sulfoxidation products 79 were observed in urine as a result of fluorine displacement, whereas 76 and a proposed acyl glucuronide of 5 were detected in bile. The formation of 76 and fluoride, along with (−)-erythro-(2R,3R)-2-fluorocitric acid (24), were also observed following incubation of 5 in mouse liver cytosol.(103)
Fluoroacetamide (10) and 2-fluoroethanol (11) are both converted to 5 and therefore share some of the same downstream products of metabolism. After ip administration of 10 to rodents, 19F NMR spectra revealed the presence of 2-fluoro-N-hydroxyacetamide (78), 24, and fluoride in tissue and excreta.(103) Administration of 11 to rodents resulted in its conversion to fluoroacetaldehyde (6), which was also detected in the liver microsomal fraction, and then on to 5 and fluoride, both which were recovered in urine. On the other hand, ip administration of 24 and (+)-erythro-(2R,3R)-2-fluorocitric acid (27) to rats resulted in their excretion mostly intact, with increasing amounts of fluoride evident in urine only after 6 h. The administration of 5 and 24 to rodents also resulted in elevated levels of citric acid (19) and increased glucose and diminished urea concentrations, suggesting disruption of the Krebs cycle and ammonia metabolism that are consistent with their known mechanisms of toxicity. The metabolic pathways of these fluorinated small molecules in rodents are summarized in Scheme 13, which also captures insights gleaned from in vitro experiments to identify the enzyme or subcellular fraction involved in each step.(103)
Scheme 13
Scheme 13. Metabolic Pathways of 5, 10, and 11 in Rodents
2. Fluorinated Alkanes
ARTICLE SECTIONS
2.1. Hydrochlorofluorocarbons and Hydrofluorocarbons
Perhalogenated alkanes or chlorofluorocarbons (CFCs) possess unique chemical and physical properties, rendering them useful as refrigerants, blowing agents, cleaning fluids, and propellants.(104) These CFCs, which contain up to three carbon atoms that are fully substituted with chlorine and fluorine atoms, are each characterized by a low boiling point, a low surface tension and viscosity, a high vapor density, and limited flammability. In addition, they have a low propensity to undergo metabolic activation leading to adverse effects on health. However, their widespread use began to be phased out in the 1990s because of their potential to absorb ultraviolet radiation and photolytically release chlorine into the stratosphere, leading to the depletion of the ozone layer.(104) To avoid these environmental drawbacks, hydrochlorofluorocarbons (HCFCs) were investigated as replacements for CFCs. HCFCs feature C–H bonds that can be oxidized in the troposphere before the molecule rises into the stratosphere.(105) HCFCs are more susceptible to metabolic activation than CFCs, generating reactive intermediates via pathways that may be associated with a variety of pathogenic consequences. In addition, many inhalation anesthetics are HCFCs and are prone to bioactivation. In this section, the biotransformation and downstream adverse effects of the more common HCFCs are summarized.
A P450-mediated oxidative dehalogenation reaction to yield trifluoroacetyl halides is common across several HCFCs, including HCFC-123 (80),(106,107) HCFC-124 (81),(108) and HCFC-125 (82).(105) These HCFCs are characterized by two halogen atoms and a single hydrogen atom bound to the same carbon atom. The rate of production of trifluoroacetic acid (67) has been demonstrated to be tightly correlated with ΔHact, the enthalpy of hydrogen atom abstraction from the carbon atom, with the energetic barrier to abstraction influenced by the number of halogen substituents present and inversely associated with their electronegativities.
HCFCs bearing one halogen and two hydrogen atoms at a single carbon are metabolized differently. Following administration of HCFC-131a (83), HCFC-132b (84), or HCFC-133a (85) to rats, the metabolite profiles in urine varied as a function of the fluorine substitution pattern (Scheme 14).(109,110) P450 catalyzed the initial oxidation of the chloromethyl carbon atom, leading to the release of HCl and the formation of a trihaloaldehyde intermediate. (The potential reproductive effects of trihaloaldehydes have been described previously in the context of trifluoroacetaldehyde (71); vide supra.) This intermediate was then partitioned among three discrete metabolic pathways. The trihaloaldehyde could be oxidized to a trihaloacid or reduced to a 1,1,1-trihaloethanol, which was subject to glucuronidation and sulfation. The halogenated ethanes 83 and 84 were ultimately converted to the carboxylic acids and glucuronidated alcohols, which were observed as the most abundant metabolites, while the sulfate conjugate was produced as a minor metabolite. In contrast, 85 was oxidized to 71, which was readily hydrated or reacted with urea (with or without elimination of water). Release of fluoride from the fluorodihalomethyl group was detected during the metabolism of all three of these halogenated ethane derivatives, although the enzymes responsible were not identified. Additionally, the conversion of 85 to aldehyde 71 in turn produced stable adducts with endogenous nucleophiles that were excreted in urine, as suggested by 19F NMR studies.
Scheme 14
Scheme 14. Metabolic Pathways of HCFCs 83–85a
aThe metabolism of 83–85 proceeds in each case through an aldehyde intermediate, which is subject to differential metabolism depending on the pattern of halogen substitution (X′, X″, and X‴).
Although HCFCs typically exhibit a favorable acute safety profile, long-term inhalation of HFC-245fa (86) (Scheme 15) led to an increase in the incidence of myocarditis in rats, a rare consequence not observed with other hydrofluorocarbons (HFCs) and likely linked to biotransformation.(111) Studies with 86 revealed a unique C–C bond cleavage reaction as a major metabolic outcome, and certain products formed along the proposed pathway may have the potential to subtend toxicity (Scheme 15).(112) Following inhalation by rats, 86 was oxidized at C-3 by P450 enzymes to afford 1,1,1,3,3-pentafluoro-1-propanol (87), which was excreted in urine. Alternatively, 87 degraded by the elimination of HF to afford the acyl fluoride 88, which was readily hydrolyzed to 3,3,3-trifluoropropanoic acid (89), another urinary metabolite. GC–MS and 19F NMR analyses also suggested the presence of multiple additional minor metabolites, including 1,1,1,3,3-pentafluoro-2-propanol (90), formed via oxidation of C-2, its oxidation product 1,1,1,3,3-pentafluoro-2-propanone (91), and the hydrate 92. Surprisingly, the main product in urine was trifluoroacetic acid (67), which was formed by a pathway that could be fully recapitulated in RLM. The product of C–C scission was proposed to follow from the interaction of 91 with the P450 Fe–hydroperoxy intermediate to afford 93 via a Baeyer–Villiger rearrangement. The ester 93 is a reactive species that can be converted to 67 enzymatically or by simple hydrolytic degradation.(113) Although covalent modification of proteins by reactive acyl fluorides is well-established, 88 was not implicated in the myocarditis induced by 86 because of its low abundance. Moreover, there is little P450 activity in cardiac tissue, and the short half-life of 88 would limit its ability to reach the heart if formed remotely. Other stable metabolites, such as 67 and 90, have been suggested to cause toxicity through distinct mechanisms, although evidence to support these hypotheses is lacking.
Scheme 15
Scheme 15. Metabolism of HFC-245fa (86)
2.1.1. Anesthetics
Halothane (94), an anesthetic implicated in adverse outcomes, including rare but severe hepatic necrosis, represents a classic example of metabolic activation leading to immune-mediated sequela and target organ toxicity.(114−118) In humans, about 80% of 94 was eliminated unchanged, with the remainder being metabolized. As summarized in Scheme 16, P450 enzymes catalyzed two distinct metabolic pathways, one reductive (path a) and the other oxidative (path b), both of which produced chemically reactive intermediates.(119) The oxidative pathway (path b) resulted in hydroxylation of the carbon atom bearing the single hydrogen atom, yielding an unstable halohydrin intermediate that collapsed by elimination of HBr. This reaction produced the highly reactive acid chloride 97, which was readily hydrolyzed to trifluoroacetic acid (67). Following administration of 94 to patients, products of this pathway were detected in urine, with 67 serving as a clinical marker of drug exposure. The reductive pathway (path a) occurred under anaerobic conditions and entailed two steps, the first being one-electron reductive removal of the bromine atom to produce a 1-chloro-2,2,2-trifluoroethyl radical.(120−122) This radical intermediate, which bears some resemblance to the trichloromethyl radical generated during the one-electron reduction of CCl4, was postulated to react with polyunsaturated lipids, leading to the release of 1-chloro-2,2,2-trifluoroethane (95), which was exhaled.(123) Additionally, this radical inactivated P450 enzymes by reaction with FeII in the active site to form an iron−σ-alkyl complex. The second metabolic step was another one-electron reduction of the radical to afford an unstable carbanionic intermediate that decomposed with the loss of fluoride to form 2-chloro-1,1-difluoroethylene (96), which was then exhaled. Studies have suggested that the hepatotoxicity induced by 94 was the result of covalent modification of hepatic proteins, leading to immune-mediated reactions and hepatic necrosis in genetically predisposed subjects upon repeated exposure to the anesthetic. In the most severe cases, this reaction was fatal. While both 97 and the 1-chloro-2,2,2-trifluoroethyl radical may have the capacity to react with nucleophilic cellular macromolecules, several lines of evidence support the involvement of acid chloride 97 in the genesis of immune-mediated toxicity. The first line of evidence was the observation of covalent binding of [14C]-labeled 94 to liver proteins, which resulted in the appearance of neoantigens in experimental animals.(124,125) Second, hepatic proteins in guinea pigs became trifluoroacetylated during exposure to 94.(126−128) Third, serum immunoglobulin G antibodies from patients with hepatitis induced by 94 were found to cross-react with hepatic neoantigens exhibiting the trifluoroacetyl group that were collected from the livers of rats and rabbits exposed to 94.(129) Serum antibodies from the same patients also recognized hepatic neoantigens from patients who died of cardiac failure following anesthesia with 94, whereas serum antibodies from control subjects did not.(130)
Scheme 16
Scheme 16. Metabolic Activation of 94, Which is Metabolized by P450 along (a) Reductive and (b) Oxidative Pathwaysa
a(a) The reductive pathway involves a one-electron reduction with loss of bromine to afford a radical, which either abstracts a hydrogen atom prior to exhalation or is further reduced. The product of reduction is an unstable 1-chloro-2,2,2-trifluoroethyl carbanion intermediate that eliminates fluoride to give 96, which is also exhaled. (b) The oxidative pathway involves oxidation and loss of HBr to give 97, which is readily hydrolyzed to give 67.
Sevoflurane (1,1,1,3,3,3-hexafluoro-2-(fluoromethoxy)propane, 98) is a polyfluorinated ether that has been popular as an inhaled anesthetic since 1990 on the basis of its rapid uptake relative to both halothane (94) and isoflurane (99).(131)
The rapid elimination of 98 through exhalation and metabolism is governed by its poor aqueous solubility, differentiating it from highly lipid-soluble anesthetics such as methoxyflurane (100). While the majority of the dose of 98 administered to humans was exhaled unchanged, approximately 2–5% underwent oxidation of the fluoromethyl group by CYP2E1, resulting in loss of fluoride to afford the formate ester 101, which was converted to hexafluoroisopropanol (HFIP, 102), as summarized in Scheme 17.(132−134) Replacement of the fluoromethyl H atom with D mitigated the formation of 102, implicating the oxidation of the fluoromethoxy moiety as the rate-limiting step in metabolism. Moreover, pretreatment of rats with EtOH prior to the preparation of liver microsomes resulted in a 3-fold increase in metabolic turnover, confirming the reliance on CYP2E1. Once formed, 102 rapidly formed the glucuronide 103, which was the main metabolite of 98 in urine. Thus, 98 is differentiated from other anesthetics like 94 and 99 by the limited extent of metabolism and the avoidance of any acyl halide products.(135−137)
The rapid elimination of 98 through exhalation and metabolism is governed by its poor aqueous solubility, differentiating it from highly lipid-soluble anesthetics such as methoxyflurane (100). While the majority of the dose of 98 administered to humans was exhaled unchanged, approximately 2–5% underwent oxidation of the fluoromethyl group by CYP2E1, resulting in loss of fluoride to afford the formate ester 101, which was converted to hexafluoroisopropanol (HFIP, 102), as summarized in Scheme 17.(132−134) Replacement of the fluoromethyl H atom with D mitigated the formation of 102, implicating the oxidation of the fluoromethoxy moiety as the rate-limiting step in metabolism. Moreover, pretreatment of rats with EtOH prior to the preparation of liver microsomes resulted in a 3-fold increase in metabolic turnover, confirming the reliance on CYP2E1. Once formed, 102 rapidly formed the glucuronide 103, which was the main metabolite of 98 in urine. Thus, 98 is differentiated from other anesthetics like 94 and 99 by the limited extent of metabolism and the avoidance of any acyl halide products.(135−137)Scheme 17
Scheme 17. Metabolism of 98
Upon contact with soda lime, which is used to absorb CO2 from breathing gases during general anesthesia, 98 is subject to base-mediated loss of HF to afford the fluorinated olefin 2-(fluoromethoxy)-1,1,3,3,3,-pentafluoro-1-propene (104), which is also known as compound A (Scheme 18).(138,139) In rats, 104 has been shown to cause nephrotoxicity, although this outcome has not been confirmed in human subjects.(139) In rats and humans, 104 was converted by GST to the saturated GSH conjugate 105, a metabolic process analogous to that observed with other fluorinated alkenes (vide infra).(140−142) Alternatively, the GSH conjugate underwent elimination to form the unsaturated GSH conjugate 106. Both conjugates were subject to sequential degradation by processes catalyzed by γ-glutamyltransferase and dipeptidases, generating the corresponding cysteine conjugates 107 and 108, respectively. The cysteine conjugates were substrates for β-lyase, which catalyzed the formation of thiolates 109 and 110. At this point, the metabolic fate of these compounds converged, with 109 losing HF and 110 simply tautomerizing, each affording 111. In the final steps, hydrolysis of 111 gave 112, which degraded further to give trifluorolactic acid (113). The GSH-dependent metabolism of 104 resembled the bioactivation of other fluorinated olefins to nephrotoxic thiolate intermediates. Surprisingly, the nephrotoxicity of 104 was exacerbated by administration of the γ-glutamyltransferase inhibitor acivicin and the β-lyase inhibitor aminooxyacetic acid, calling into question the role of this pathway in the onset of toxicity.(143,144) However, no alternative pathway for the metabolic activation of 98 has been proposed.
Scheme 18
Scheme 18. Metabolism of 104, a Base-Catalyzed Degradation Product of 98, in Rats and Humans
2.2. Drug-like Compounds Featuring Fluorinated and Difluorinated Alkanes
Under physiological conditions, both biologic nucleophiles and drug-metabolizing enzymes can participate in reactions that cleave the C–F bonds of monofluoroalkyl groups to liberate fluoride.(27) Such reactions have been observed in studies of [18F]-labeled PET tracers, since the fate of the radioactive fluoride can be easily tracked. As a PET tracer, the [18F] radionuclide offers certain advantages.(145) First, the radiological half-life (110 min) is comparable to the typical length of the imaging window, minimizing the radiation burden for the patient and enabling repeat imaging on successive days. Second, networks for the distribution of [18F]fluorodeoxyglucose have demonstrated that production of [18F]-labeled radiopharmaceuticals at central sites is a reasonable alternative to on-site production.(146) Thus, the [18F] isotope has become the preferred radionuclide for PET imaging studies. Procedures to install [18F] into organic molecules must proceed rapidly and with high radiochemical yield to be useful, and the number of suitable reactions is increasing.(15−20) One such reaction involves the displacement of a good leaving group by [18F]fluoride at an aliphatic or aromatic position. However, since it is often difficult to incorporate an [18F] label into an aromatic compound in the final synthetic step, the addition of a larger appendage, such as an aromatic ring incorporating a [18F]fluoroalkyl substituent, is used as an alternative approach.(147) To assist in the development of this approach, the fate of fluoroalkyl groups appended to a phenyl ring was examined using human serum and RLM as proxies for stability in vivo.(148) The three homologous 4-(ω-[18F]fluoroalkyl)biphenyls 114, 117, and 121 depicted in Scheme 19 were prepared as model compounds, and all eliminated [18F]fluoride to some degree under the test conditions. In human serum, [18F]fluoroethylbiphenyl 117 and [18F]fluoropropylbiphenyl 121 were relatively stable, with more than 90% remaining after 1 h. In contrast, [18F]-fluoromethylbiphenyl 114 was converted extensively (40 to 50% degraded over 3 h) to [18F]fluoride and the benzylic oxidation product 115, which would convert spontaneously to [1,1′-biphenyl]-4-carbaldehyde (116).(148) Similar trends were observed in RLM, in which 114 suffered from increased reactivity relative to 117 and 121, suggesting that this motif should be avoided for PET imaging studies. Moreover, this degree of chemical reactivity could be interpreted as a safety issue since biologic targets bearing a proximal thiol or other nucleophile may become alkylated, advocating for caution when exploiting electronically activated fluorides.(27)
Scheme 19
Scheme 19. In Vitro Metabolism of a Homologous Series of [18F]fluoroalkylbiphenyl Derivativesa
aBenzylic oxidation of 114 releases fluoride immediately, whereas 117 and 121 require successive benzylic oxidations via the intermediate alcohols 118 and 122, respectively, to afford the ketones 119 and 123. Since the release of fluoride is faster from the intermediate derived from 121 than that from 117, the latter is deemed to have the best overall properties. This compound is oxidized in successive steps to 118 and the α-fluoro ketone 119, which is believed to undergo spontaneous but slow α-elimination (16% over 1 h) to give the corresponding enol. This enol is tautomeric with the ketone 120.
A number of estrogen analogues bearing 11β-(ω-fluoroalkyl) and 11β-(ω-fluoroalkoxy) moieties, represented generically by 125 and 126, respectively, have been explored for their potential to function as PET agents for imaging of estrogen-receptor-positive breast cancers (Figure 5).(149) Most were characterized by selective distribution in female rats, with 10- to 40-fold higher concentrations in the uterus compared with the blood or muscle. The chain length and choice of a carbon or oxygen linkage had implications for both the estrogen receptor binding affinity (improved for 11β-(ω-fluoroalkyl) derivatives 125 compared with the 11β-(ω-fluoroalkoxy) estrogens 126) and the extent of distribution of radioactivity into the bone, which was taken as a proxy for the degree of metabolic defluorination. 11β-(2-Fluoroethoxy)estradiol (128) was the least prone to defluorination, whereas 11β-(2-fluoroethyl)estradiol (127) was the most susceptible.(149) This trend is perhaps surprising since the placement of fluorine in the former molecule does not directly block a site of hydrogen atom abstraction; however, the result does recall a trend observed elsewhere that fluorine substitution at the distal end of a short alkoxy chain can decrease the rate of O-dealkylation.(150) Consistent with this observation, fluorinated alkyl groups have been found to confer protection against oxidation at remote sites.(151)
Figure 5
Figure 5. 11β-Fluoroalkyl and 11β-fluoroalkoxy estrogen derivatives explored for their potential as PET imaging agents.
11C-WAY-100635 (11C-129) has been used to image central serotonin 5-HT1A receptors and monitor serotonin-related neuronal activity (Figure 6).(152,153) However, the installation of the 11C label at the carbonyl carbon of 129 requires a complex synthetic sequence that carries an approximately 10% failure rate. The short radiological half-life of 11C (20 min) also limits the use of 11C-129 to hospitals equipped with an in-house cyclotron and radiopharmacy. In light of these drawbacks, efforts have been made, with mixed success, to identify [18F]-labeled analogues of 129 that could be used as alternatives. One of these compounds, MPPF (130), was characterized by lower 5-HT1A selectivity and lower rat brain uptake, while another, FCWAY (131), underwent defluorination in humans, resulting in PET images of poor quality. PET imaging studies with the three [18F]fluoromethyl bridge-fused ring analogues 133–135 demonstrated specific binding to brain regions rich in 5-HT1A receptors in rats.(154) However, these studies also found high rates of radioactivity uptake into bone indicative of defluorination, an outcome that was not forecasted by chemical stability studies using conditions that favored the elimination of HF. Whereas the proclivity of these compounds toward defluorination in vivo would prevent their practical application in human PET imaging, a related compound, [18F]-MEFWAY ([18F]-132), exhibits improved stability in rats and offers continued potential in this area (Figure 6).(155)
Figure 6
Figure 6. Structures of 11C-WAY-100635 (11C-129) and [18F]-derived analogues 130–135 explored as potential PET tracers. The asterisk denotes the position of the 11C label in 11C-129.
Vicinal difluorinated alkanes have found infrequent use in drug design, although they exhibit favorable physicochemical attributes and appear to offer a degree of metabolic stability based on studies using Caenorhabditis elegans.(156−158) The oxidation pathways observed in one such study of all-cis polyfluorinated cyclohexane derivatives primarily targeted methylene and benzylic groups that were not substituted by fluorine. The lone exception was observed during the metabolism of ((1R,2S,3R)-2,3-difluorocyclohexyl)benzene (136), which gave rise to the simple hydroxylated derivatives 137–139 along with the desfluoro metabolite 140 (Scheme 20).(156) This reaction mechanism was not rationalized and ostensibly involved the oxidation of a remote methylene. The study also noted certain physicochemical trends in this series, including an increase in polarity with increasing fluorination, as measured by a reduction in log P values, and a preference for H2O to associate with the hydrogen-bearing face opposite to that projecting the cis-oriented fluorine atoms. The latter was attributed to the strong polarization of the hydrogen atoms by the adjacent fluorine substituents.(156)
Scheme 20
Scheme 20. Metabolism of ((1R,2S,3R)-2,3-Difluorocyclohexyl)benzene (136) by C. elegansa
aComplete conversion was obtained over 72 h at 28 °C. Compounds 137–139 represent the metabolites observed in the study, which included other polyfluorinated substrates in addition to 136. The main site of oxidation was at the benzyic carbon rather than at sites substituted by fluorine. The unexpected formation of 140 was not explained but serves as a reminder that fluorinated alkyl groups may not always be metabolically inert.
The selection of a geminal or vicinal difluorinated alkane in drug design will depend on several factors, including effects on lipophilicity and solubility, properties that are readily calculated in silico.(158) Of particular importance, the molecular volume and the solvation energy of the fluorinated compounds and the overall dipole moment of the fluorinated fragment can be approximated reasonably well by summing the dipole moments of the individual C–F bonds. In this regard, vicinal difluoro groups can be judiciously deployed to help improve drug-like properties.(158) In the preferred gauche conformational arrangement, a vicinal difluoroalkane adopts a F–C–C–F dihedral angle of approximately 70°, which is smaller than the 104° angle adopted by the C–F bonds of a geminal difluoro group. Since the former angle is smaller, the sum of the polarity vectors is longer, resulting in a larger net dipole moment. This increased polarity contributed to an overall effect of increased solubility and reduced lipophilicity for a vicinally difluorinated compound compared with a geminally difluorinated isomer, with no appreciable difference between the molecular volumes of the two compounds.(25,158) This trend was illustrated by the measured properties of the matched pair of difluoroalkyl indoles 141 and 142 as well as by a comparison of the matched pair of hypoxia-inducible factor-2α (HIF2α) inhibitors 143 (PT2385) and 144 (PT2977), both of which were advanced into clinical trials for the treatment of advanced clear cell renal cell carcinoma (Figure 7).(159,160) The metabolite profile of 143 included metabolites formed by oxidative defluorination of the fluorophenyl ring and, separately, glutathione conjugation of the same ring.(159) While no metabolites that represented modifications to the gem-difluoro moiety were identified, 143 suffered from extensive glucuronidation that resulted in suboptimal pharmacokinetics in humans.(159) These shortcomings were corrected by the cis-vic-difluoro isomer 144, which was characterized by decreased lipophilicity, lower protein binding, and attenuated glucuronidation, perhaps as a result of the diminished acidity of the proximal cis-hydroxyl group.(160)
Figure 7
Figure 7. Influence of the pattern of difluorination on the physicochemical properties of the matched pair of indoles 141 and 142 and the HIF2α inhibitors 143 (PT2385) and 144 (PT2977). The solubilities of 141 and 142 were measured in aqueous buffer (pH 6.5), while clearance was measured in human liver microsomes and protein binding of 143 and 144 was measured in human plasma.
The fluorinated allylic position of fluticasone furoate (145) was susceptible to hydroxylation by P450, resulting in the loss of fluorine.(161) While a specific mechanism for the oxidation of 145 has not been proposed, mechanistic studies of the metabolism of the closely related corticosteroid flunisolide (146) (Scheme 21B) suggest the pathway presented in Scheme 21A.(162,163) On the basis of its bound conformation, 145 is primed for 6-β-hydroxylation, since the fluorine substituent occupies the equatorial position and the axial hydrogen is exposed for abstraction by the enzyme.(164) Following rebound of the hydroxyl radical, the fluoroalcohol 147 collapses to the keto intermediate 148, which is conjugated to the 2-carbonyl via the double bond at the 4,5-position. The ketone is then reduced to the observed metabolite 149. While the stereochemistry of the product has not been determined, the corresponding product of flunisolide metabolism that was produced via 150 and 151, 6-OH-desfluoroflunisolide (152), was believed to adopt the R conformation at the site of metabolism (Scheme 21B). Similarly, non-fluorinated steroids like testosterone are hydroxylated on the β face.(165) Thus, the hydroxyl substituent in 152 is installed with the same axial configuration as the hydrogen abstracted from 146.
Scheme 21
Scheme 21. Fluticasone Furoate (145) and Flunisolide (146) Are Both Metabolized by Allylic Oxidation at C-6, Which Results in the Loss of Fluorinea
a(A) Fluticasone furoate (145) undergoes abstraction of the axial 6-hydrogen by P450 with rebound oxidation, leading to 147. This intermediate eliminates fluoride to form the keto compound 148, which is reduced to the 6-hydroxy metabolite 149. While the configuration of 149 has not been determined, analogy to flunisolide (146), which is metabolized in a stereospecific manner (B), would suggest that both steroids likely undergo β-oxidation at this site.
Simpler allylic fluorides lacking the activation afforded by the carbonyl moiety of 145 have also been observed to undergo hydrolytic cleavage of the C–F bond in mouse liver microsomes.(166)
3. Fluorinated Alkenes
ARTICLE SECTIONS
3.1. Volatile Fluorinated Alkenes
Fluorinated alkenes can mimic the topology and physicochemical properties of ketones, esters, and amides (Figure 8a) and carbonyls (Figure 8b), providing improved metabolic stability and potency.(1−3) The fluoroalkene retains features of the amide bond of peptides, including bond length, bond angle, and a significant dipole moment, while permitting either an E or Z configuration. Additionally, the electronegativity of fluorine imbues the alkene with electronic properties that are similar to those of the carbonyl group with respect to dipole moment, charge distribution, and electrostatic potential. Hence, the replacement of amides by fluoroalkenes offers certain advantages, such as resistance to hydrolysis, while retaining desirable steric and electronic characteristics.(1−3) These features have been employed in the design of inhibitors of dipeptidyl peptidase IV (DPP4), thermolysin, and the HCV NS5A replication complex cofactor.(167,168) In peptidomimetic design, fluoroalkenes have been explored as peptide-bond surrogates to amplify or attenuate chemical properties and obtain a deeper understanding of the structure and function of a peptide or protein. For example, the influence of the prolylamide bond geometry on the inhibition of DPP4 has been investigated.(169)
Figure 8
Figure 8. Fluorinated alkenes can serve as isosteres of a ketone, ester, or amide (A) or a carbonyl moiety (B), depending on the specific structural arrangement.
While fluorinated alkenes have been explored broadly in medicinal chemistry, limited information is available on their metabolic pathways. However, fluorinated alkene moieties are found in HFCs and perfluorocarbons, and studies of these compounds have provided some insight into their metabolic fate. HFCs consisting of only carbon, fluorine, and hydrogen atoms were designed as refrigerants with lower greenhouse-gas potency relative to ozone-depleting refrigerants such as 1,1,1,2-tetrafluoroethane (153) and CFCs such as trichlorofluoromethane (154).(104) Among the marketed fluorinated refrigerants are 2,3,3,3-tetrafluoropropene (155) and 1,3,3,3-tetrafluoropropene (156). The environmental advantage of 155 stems from its short atmospheric lifetime of approximately 11 days, which is a significant improvement over that of 153 (14.6 years), and its decomposition to trifluoroacetic acid (67), which is subject to wet and dry deposition.(170,171) On the other hand, perfluorocarbons have been used to produce fluorinated carbon chain polymers that are components of household and commercial products such as waterproofing agents, lubricants, and sealants.
Fluorinated alkenes caused varying degrees of acute toxicity in rats following 4 h of inhalation, with LD50 values ranging from 40 000 ppm for 157 and 3000 ppm for 158 to <1 ppm for 159.(172) The observed toxicity coincided with the inherent electrophilicity of these molecules, with the lesser-substituted termini of 158 and 159 predisposing toward nucleophilic substitution at these sites.(172) The addition of a nucleophile to 157–159 gave rise to carbanionic intermediates that eliminated fluoride to restore an olefin moiety (Scheme 22). The reactivity of the double bonds of 157–159 increased in a fashion commensurate with the stability of the proposed carbanion intermediates (159 > 158 > 157), as captured in Figure 9.(172−174) In the case of 158 and 159, elimination of fluoride from the intermediate carbanion (163) afforded conjugates where the biological nucleophile became substituted with an allyl moiety (165; Scheme 22, path b, purple) rather than a vinyl moiety (164; Scheme 22, path a, red). Therefore, the rank order of the acute toxicities (LD50 values) of these perfluorinated olefins in rats suggested a chemical reaction with biological macromolecules as a key step underlying the adverse effects.
Scheme 22
Scheme 22. Proposed Nucleophilic Substitution of 1,1,3,3,3-Pentafluoro-2-(trifluoromethyl)prop-1-ene (158) with Nucleophiles
Figure 9
Figure 9. Correlation among the reactivity of perfluorinated olefins, the stability of carbanion intermediates, and lethality.
The intrinsic chemical reactivity of perfluorinated alkenes toward nucleophiles may explain the lethality observed in acute inhalation studies. However, long-term toxicology studies of fluorinated olefins that are characterized by low acute toxicity have revealed that the differentiated effects on tissues and cellular compartments (e.g., genotoxicity, cytotoxicity, hepatotoxicity, nephrotoxicity) can be attributed to other metabolic pathways. Among the fluorinated alkenes, vinyl fluoride (160) is notable because of its use as a monomer in the production of polyvinyl fluoride and other fluoropolymers.(175) The primary safety concern with 160 is occupational exposure via inhalation that exceeds the maximum levels recommended over intervals of 8 h (1 ppm) or 15 min (5 ppm).(176,177) In cancer bioassays, inhalation of 160 produced exposure-dependent hepatic, pulmonary, and Harderian gland tumors in rats and hepatic and Zymbal gland tumors in mice.(176) The spectrum of carcinogenic effects associated with 160 in laboratory animals was similar to those of vinyl chloride (161) and vinyl bromide (162), a known and a probable human carcinogen, respectively, implicating 160 as a probable human carcinogen.(177−179) Multiple observations from mutagenicity and clastogenicity studies have been used to establish the mechanisms underlying carcinogenicity induced by 160.(176) Exposure to 160 was associated with dose-dependent mutagenicity in Salmonella typhimurium (strain TA1535) and caused hprt forward mutations and chromosomal aberrations in Chinese hamster ovary cells in the presence of Aroclor-1254-induced rat liver S9.(176) However, the results of cytogenetic assays in rats were equivocal, with no DNA strand breaks or cross-links found.(176)
Since 160 was mutagenic upon metabolic activation in susceptible bacteria, a mechanism was proposed that involved the formation of chemical intermediates that would react with genetic material. The postulated mechanism, which was informed by the known chemical toxicology of 161 and 162, involved oxidation of 160 to fluoroethylene oxide (166) followed by conversion to fluoroacetaldehyde (6) and fluoroacetic acid (5) (Scheme 23).(180,181) The enzyme responsible for the initial epoxidation was CYP2E1, which is expressed in the target tissues (e.g., liver, lung) of nonclinical species and is known to prefer small xenobiotics as substrates. Inhibition (by 4-methylpyrazole) and induction (by EtOH) either blocked or enhanced the metabolism of 160, respectively, in agreement with this hypothesis.(182) In addition, the hepatotoxicity of 160 in rats was exacerbated upon coadministration of trichloropropylene oxide, an inactivator of epoxide hydrolase that would interfere with detoxification of 166 by hydrolysis, further supporting the importance of this fluorinated epoxide in the onset of toxicity.(183)
Scheme 23
Scheme 23. Metabolic Activation of 160 by CYP2E1 to Generate Fluoroethylene Oxide (166), a Reactive Intermediate That Can Modify DNA Bases or Form Downstream Products
Epoxide 166 is characterized by inherent ring strain, while its intrinsic electrophilicity is enhanced by the electron-withdrawing fluorine substituent. This set of circumstances facilitated the reaction of 166 with nucleophilic nitrogen atoms of DNA bases, a reaction that led to the formation of 7-(2-oxoethyl)guanine (173), a product of DNA alkylation that was observed in rats following exposure to the related olefin 161 (Scheme 23).(180,184) Another outcome involved adduction of the primary amine moiety leading to 169, which degraded to 170 and then cyclized to 171. Dehydration of 171 produced N2,3-ethenoguanine (172), which can cause miscoding mutations characterized by GC to AT transitions.(185,186) Rodents exposed to 160 (up to 2500 ppm for 12 months) exhibited levels of hepatic 172 that correlated positively with tumor incidence (hemeangiosarcoma), further demonstrating the quantitative relationship between modification of genetic material by 160 intermediates and genotoxic outcome.(187,188) Although 6 could theoretically react with the nucleic acids to form adducts, studies of 160 have implicated the epoxide intermediate 166 in DNA alkylation leading to mutagenic effects.(186)
In addition to genotoxicity, studies in rats indicated that administration of 160 increased acetyl-CoA levels and exhalation of acetone, suggesting incorporation of 5 into the citric acid cycle and disruption of energy metabolism.(189) Moreover, incubation of 160 in RLM resulted in N-alkylation of the porphyrin ring of P450 enzymes, rendering them inactive.(190) The modification of the heme moiety was likely initiated by a free radical intermediate derived from 160.
Whereas 160 was mutagenic following metabolic activation by P450, its perfluorinated analogue 157 caused renal toxicity, but not hepatotoxicity, via a distinct metabolic route.(191,192) Since 157 is a key monomer for the production of polytetrafluoroethylene (Teflon) and other polymers, its toxicity in laboratory animals is well-documented.(193) The nephrotoxicity of 157 manifested as dose-dependent renal tubule karyomegaly, which was evident in mice exposed to 157 at more than 1250 ppm over 15 and 90 days.(194) Metabolic activation of 157 was independent of P450, as suggested by the finding that inhibition of the monooxygenases by CO did not affect its metabolism.(191) Instead, the double bond of 157 was subject to direct GSH conjugation by hepatic microsomal GST in rats.(191) Although the primary addition product S-(1,1,2,2-tetrafluoroethyl)GSH (174) was absent from bile, its downstream products, specifically S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (175) and a minor cysteinylglycine conjugate, were detected, suggesting rapid processing of 174 by γ-glutamyl transpeptidase and dipeptidase in bile canaliculi or hepatocytes (Scheme 24). It is believed that 175 is reabsorbed and further metabolized by renal β-lyase to generate a reactive and cytotoxic product, 1,1,2,2-tetrafluoroethane-1-thiolate (176) along with pyruvic acid and NH3. Direct evidence linking 175 to nephrotoxicity was obtained in experiments that recapitulated the adverse renal effects of 157, including renal cell proliferation and histopathological changes in the tubules, following oral administration of synthetic 175 to rodents for 12 days.(191,192) The role of renal uptake of 175 in the expression of nephrotoxicity was further confirmed by the observation that coadministration of 175 with the renal anion transporter inhibitor probenecid blocked the toxicity in rats. While the ultimate toxicant in this example remains unknown, it is postulated that 176 may suffer loss of fluorine to give 177, an electrophilic thionoacyl fluoride derivative capable of thioacylating cellular macromolecules.(195) Similar findings have been reported during the study of other nephrotoxic haloalkenes, including chlorotrifluoroethylene (178), 1,1-dichloro-2,2-difluoroethylene (179), 1,1-dibromo-2,2-difluoroethylene (180), and hexachlorobutadiene (181).(196−199) Additional mechanistic studies have suggested that the nephrotoxicity of 175 may be exacerbated by an additional effect on ion transport.(191)
Scheme 24
Scheme 24. Proposed Metabolic Pathway of 157 Leading to Tissue-Selective Toxicitya
aThe reaction of 157 with hepatic GSH generates S-(1,1,2,2-tetrafluoroethyl)GS (174), which is processed to S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (175) and excreted into bile. Following absorption, 175 undergoes β-lysis to form the thiolate intermediate 176 along with pyruvic acid and ammonia. Elimination of fluoride from 176 generates the reactive thionoacyl fluoride 177, which is hypothesized to be the ultimate toxicant.
The first several steps of the GSH-dependent metabolic modification of 2-bromo-2-chloro-1,1-difluoroethylene (182), a decomposition product of halothane (94), mirrored those of 1,1-difluoroalkenes (Scheme 25). This compound was metabolized to the cysteine conjugate 183, which was converted by β-lyase to the ethanethiolate 184, a molecule that eliminated F– to give 185. It was postulated that 185 underwent a series of reactions that involved dehalogenation, hydrolysis, and loss of H2S to form glyoxylic acid (191), the ultimate product of the metabolism of 182 in rat kidney homogenates and in vivo.(195,196)
Scheme 25
Scheme 25. Metabolism of 2-Bromo-2-chloro-1,1-difluoroethylene (182)
3.2. Fluorinated Alkenes as Mechanism-Based Enzyme Inhibitors
In addition to their uses as refrigerants and polymer precursors, fluorinated alkenes (monofluoro or gem-difluoro derivatives) have been employed in the design of MBIs of several enzymes.(200) These irreversible inhibitors, although chemically inert in solution, typically mimic the native substrate of the target enzyme and undergo metabolic activation to an electrophile that reacts with the enzyme or its cofactor. To inactivate a target enzyme selectively, these electrophiles must react prior to diffusing from the active site. Consequently, the intrinsic chemical reactivity of the electrophile is a key consideration in their design. Fluorine has proven useful as a leaving group in this context, with its diminutive size minimizing steric interference and facilitating substrate mimicry and recognition by enzymes.(200) These features represent advantages of fluorine relative to other halogen substituents. This section describes the design and postulated mechanisms of fluorinated alkenes as irreversible inhibitors of enzymes that are involved in a variety of biochemical processes.
Squalene monooxygenase (SM), sometimes called squalene epoxidase, catalyzes the conversion of squalene (192) to (3S)-2,3-oxidosqualene (193), the first and rate-limiting step in the biosynthesis of sterols in mammals, plants, and fungi.(201,202) This enzyme uses molecular oxygen to effect the epoxidation and requires NADPH and FAD as cofactors (Scheme 26).(203) Hence, SM is a potential therapeutic target in the design of drugs to treat fungal infections, hypercholesterolemia, and cancer.(201,202) While reversible SM inhibitors have been described, replacement of the terminal methyl groups of squalene by fluorine atoms afforded the time-dependent SM inhibitors 194 and 195.(204) Both the orientation of the difluorinated olefin and the chain length were suggested to be important for inhibition, since the sterically more congested analogues 196 and 197, which incorporate branched or elongated fluorinated olefins, did not inactivate SM. Dithiotheritol (500 μM) did not prevent inhibition by 194 or 195 in vitro, suggesting that these compounds inactivated SM without reentering solution. Although the mechanism of inhibition of these compounds was not articulated, it is reasonable to believe that 194 and 195 undergo oxidation by SM to form fluorinated epoxides that are more electrophilic than 193. Interestingly, the monofluoro derivatives 198 and 199 and the non-fluorinated compound 200 were weaker inhibitors of SM, highlighting the influence of the fluorine atoms on the potency and epoxide electrophilicity.(204)
Scheme 26
Scheme 26. Conversion of Squalene (192) to (3S)-2,3-Oxidosqualene (193) by Squalene Monooxygenase, the Initial Step in the Biosynthesis of Sterols
S-Adenosyl-l-homocysteine hydrolase (SAHase) is an NAD+-dependent enzyme that regulates transmethylation reactions in many organisms and helps maintain cellular function.(205,206) Inhibition of SAHase leads to a build-up of S-adenosyl-l-homocysteine, turning off intracellular transmethylation via a feedback loop. The broad-spectrum antiviral agent neplanocin A (201), an analogue of cyclopentenyl adenosine, is a reversible type-I MBI of SAHase (Ki = 8.39 nM) that is converted by the enzyme to the 3′-keto product 203, forcing the unproductive consumption of NAD+ (Scheme 27).(207,208) An effort to improve the potency of this compound led to the discovery of fluoroneplanocin A (202), a compound characterized by a degree of irreversible enzyme inhibition that was, surprisingly, nonstoichiometric.(209) Further analysis revealed 202 to exhibit a mixed mode of inhibition that was characterized by both reversible type-I and irreversible type-II behavior.(209) The 3′-keto variants of both inhibitors, 203 and 204, exited the enzyme, resulting in type-I reversible inhibition, and both underwent nucleophilic attack by SAHase, forming 205 and 206, respectively. However, only 206 eliminated fluoride to bind irreversibly (207; Scheme 27). This mixed mode of inhibition contributed to the improved potency observed in the case of the fluorinated homologue 202.(209)
Scheme 27
Scheme 27. Proposed Mechanisms of Inhibition of SAHase by Neoplanocin A (201) and Fluoroneplanocin A (202)
The flavin-dependent monoamine oxidases MAO-A and MAO-B catalyze the oxidative deamination of aminergic neurotransmitters and dietary amines.(210−212) Although inhibition of MAOs was explored as a means of therapeutically elevating levels of neurotransmitters in the brain, the pharmacology was complex since these isoforms are differentially expressed in tissues and differentially engaged by substrates and inhibitors. MAO-A exhibits broader substrate specificity, metabolizing both neurotransmitters (e.g., norepinephrine, serotonin, and dopamine) and dietary amines (tyramine), whereas MAO-B is mainly involved in the metabolism of dopamine and arylalkylamines.
Following the introduction of nonselective MAO inhibitors to treat depression in the 1950s, their safety was compromised by a phenomenon known commonly as the cheese effect. Inhibition of MAO-A by these medicines led to a spike in the levels of tyramine, a naturally occurring compound abundant in certain foods such as cheese, to dangerous levels that were associated with cardiovascular events and hypertension. While l-deprenyl (selegiline, 208), 
a selective and irreversible inhibitor of MAO-B used for the treatment of early-stage Parkinson’s disease, did not cause such effects at therapeutic concentrations, its plasma levels became elevated by interaction with oral contraceptives to the extent that the adverse effects related to inactivation of MAO-A were evident.(213)
a selective and irreversible inhibitor of MAO-B used for the treatment of early-stage Parkinson’s disease, did not cause such effects at therapeutic concentrations, its plasma levels became elevated by interaction with oral contraceptives to the extent that the adverse effects related to inactivation of MAO-A were evident.(213)Fluorinated inhibitors of MAO with improved isoform selectivity were investigated in clinical trials in the 1980s. As a representative of the fluorophenyl allylamine class, mofegiline (209) was developed as an irreversible inhibitor of MAO-B that exhibited good potency and 200-fold selectivity over MAO-A.(214,215) The p-fluorophenyl substituent contributed to the superior isoform selectivity over MAO-A, of which 209 was a weak and reversible inhibitor. The mechanism of inhibition of MAO-B by 209 involved oxidation of the primary amine to yield an iminium-based Michael acceptor 210 that was subject to nucleophilic attack at the terminus of the fluorinated olefin by the cofactor to afford 211, as depicted in Scheme 28. The departure of fluoride from 211 resulted in irreversible binding to the cofactor and inactivation of the enzyme (212).(214,215)
Scheme 28
Scheme 28. Inhibition of MAO B by Mofegiline (209)a
aThe inhibition results from oxidation of the primary amine to the conjugated iminium 210, a Michael acceptor that reacts with N5 of the flavin coenzyme, forming the covalent adduct 211. Loss of fluoride from 211 results in the formation of a covalent complex (212).
Mofegiline (209) also exhibited off-target effects via the inhibition of semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1), an enzyme that oxidizes primary amines to aldehydes, releasing NH3 and H2O2 in the process.(216,217) Inactivation of SSAO/VAP-1 led to an anti-inflammatory effect in preclinical species.(218) The primary amine of 209 was believed to act as a nucleophile in its interaction with SSAO/VAP-1, condensing with the cofactor 2,4,5-trihydroxyphenylalanine (213) in its quinone form 214 (topaquinone) to give the quinone imine 215 (Scheme 29). Tautomerization of 215 afforded the electrophilic conjugated imine intermediate 216. This imine reacted with the enzyme to afford 217, a compound that released fluoride to give the irreversibly bound conjugate 218. The characterization of this pathway led to the discovery of additional irreversible inhibitors of SSAO/VAP-1 that retained the fluorinated olefin and primary amine moieties but were devoid of activity against MAO B.(219)
Scheme 29
Scheme 29. Inactivation of SSAO/VAP-1 by Mofegiline (209)a
aMofegiline is believed to condense with (2,4,5-trihydroxyphenyl)alanine quinone (214), the SSAO/VAP-1 cofactor, to form the quinone imine 215. Tautomerization of 215 affords the conjugated imine 216, which is intercepted by a proximal nucleophilic amino acid residue to give 217. This intermediate can eliminate fluoride, leading to the irreversibly inactivated enzyme 218.
The pharmacokinetics and disposition of 209 were investigated in both preclinical species and humans.(220) Urine samples that were collected following oral administration of 209 contained four major metabolites, 219, 220, 222, and 223, which collectively accounted for 70% and 40% of the dose in dogs (collection interval of 0 to 24 h) and humans (0 to 8 h), respectively (Scheme 30). The initial step in the formation of both 222 and 223 was believed to be the addition of CO2 to the primary amine moiety of 209 to form the carbamic acid intermediate 221 (Scheme 30). As depicted in Scheme 31, oxidation of the fluorovinyl moiety of 221 afforded the epoxide 224, which was subject to an intramolecular nucleophilic attack by the pendent carboxylate to afford aldehyde 225 via an unstable fluorohydrin intermediate that released fluoride. Subsequent oxidation of 225, presumably by aldehyde oxidase, gave rise to the carboxylic acid 222.(220)
Scheme 30
Scheme 30. Metabolic Pathways of Mofegiline (209) in Dogs and Humans
Scheme 31
Scheme 31. Proposed Mechanism for the Formation of 222 from 209 via Carbamic Acid 221
Glucuronidation of the carbamic acid 221 by UGT provided 223, while succinylation of the primary amine function by N-succinyl transferase gave 219. The urea derivative 220, an unusual enamino aldehyde, was proposed to form via the multistep reaction depicted in Scheme 32. Completing the metabolite profile, oxidative deamination of 209 by MAO afforded the fluorinated α,β-unsaturated aldehyde 226, which reacted with H2O to give an unstable fluorohydrin intermediate. This intermediate released fluoride to give the dialdehyde derivative 227, which was subject to condensation with urea to generate the final product 220.(220)
Scheme 32
Scheme 32. Proposed Mechanism for the Formation of 220 from 209
Although fluorinated alkenes have been exploited in the design of covalent inhibitors, they can also react with enzymes in ways that might be difficult to anticipate. CPP-115 (228) 
represents an example of a fluorinated alkene that was designed as an MBI of γ-aminobutyric acid aminotransferase (GABAAT) but engaged the enzyme in an unexpected manner.(221) By way of background, GABAAT is a PLP-dependent enzyme responsible for degrading the inhibitory neurotransmitter γ-aminobutyric acid (GABA).(222) Inhibition of GABAAT leads to elevated levels of GABA in the brain and can help patients overcome seizures or substance abuse. MBIs of GABAAT have been designed that resemble the natural substrate in their interactions with PLP, with the introduction of structural modifications designed to effect covalent binding to the enzyme and interrupt enzymatic function.(222) Vigabatrin (229, γ-vinyl-GABA or 4-amino-5-hexenoic acid) remains the only such GABAAT inhibitor approved to treat infantile spasms and refractory partial seizures.(223) However, 229, which is administered at doses of 500 mg or more twice daily, has been associated with severe clinical adverse effects, including nephrotoxicity and blindness. The proposed mechanism for the reaction between GABAAT and 229 is illustrated in Scheme 33. This mechanism postulated two possible outcomes from the intermediate 230, with the major path (path a, 70%) irreversible and the minor path (path b, 30%) reversible. Path a proposed that 230 was deprotonated by the catalytic Lys329 to afford an α,β-unsaturated iminium that reacted with Lys329 to produce 231, a complex that was stable toward denaturing conditions and was characterized by X-ray crystallography.(224) Path b postulated that the olefin moiety participated in deprotonation of 230 to produce an aminal-type intermediate that degraded with release of the nucleophilic enamine 232. This enamine was poised to react with the GABAAT-Lys iminium species to give 233.
represents an example of a fluorinated alkene that was designed as an MBI of γ-aminobutyric acid aminotransferase (GABAAT) but engaged the enzyme in an unexpected manner.(221) By way of background, GABAAT is a PLP-dependent enzyme responsible for degrading the inhibitory neurotransmitter γ-aminobutyric acid (GABA).(222) Inhibition of GABAAT leads to elevated levels of GABA in the brain and can help patients overcome seizures or substance abuse. MBIs of GABAAT have been designed that resemble the natural substrate in their interactions with PLP, with the introduction of structural modifications designed to effect covalent binding to the enzyme and interrupt enzymatic function.(222) Vigabatrin (229, γ-vinyl-GABA or 4-amino-5-hexenoic acid) remains the only such GABAAT inhibitor approved to treat infantile spasms and refractory partial seizures.(223) However, 229, which is administered at doses of 500 mg or more twice daily, has been associated with severe clinical adverse effects, including nephrotoxicity and blindness. The proposed mechanism for the reaction between GABAAT and 229 is illustrated in Scheme 33. This mechanism postulated two possible outcomes from the intermediate 230, with the major path (path a, 70%) irreversible and the minor path (path b, 30%) reversible. Path a proposed that 230 was deprotonated by the catalytic Lys329 to afford an α,β-unsaturated iminium that reacted with Lys329 to produce 231, a complex that was stable toward denaturing conditions and was characterized by X-ray crystallography.(224) Path b postulated that the olefin moiety participated in deprotonation of 230 to produce an aminal-type intermediate that degraded with release of the nucleophilic enamine 232. This enamine was poised to react with the GABAAT-Lys iminium species to give 233.Scheme 33
Scheme 33. Proposed Mechanism of Irreversible (Path a) and Reversible (Path b) Inhibition of GABAAT by the GABA Mimic Vigabatrin (229)
To improve on the properties of 229, the conformationally restricted and fluorinated analogue 228 was prepared. This compound was designed to give rise to an intermediate that would be a more reactive Michael acceptor and, by extension, a more efficient inactivator of GABAAT than 228.(225) Indeed, 228 was 187-fold more potent than 229 and offered a clinical profile that suggested favorable absorption, distribution, metabolism, excretion, and safety properties.(226) However, a study of the inhibitory mechanism associated with 228 revealed some unexpected findings, including the absence of any covalent modifications to GABAAT, as evidenced by intact mass spectrometry of the protein following treatment with 228. An X-ray crystal structure of this protein revealed the orientation of the ultimate inhibitor and demonstrated that 228, although designed to inhibit GABAAT irreversibly via a Michael addition, actually engaged in a multistep defluorination pathway to yield the dicarboxylic acid 240. This acid formed tight electrostatic interactions with the two key arginine residues Arg192 and Arg445 of the enzyme and induced a conformational change in the active site (Scheme 34).(226) It was postulated that the addition of H2O to 236, rather than the catalytic Lys329 as observed with 229, was preferred since the electrophilic center was not presented in a geometrical orientation that was compatible with a productive reaction with Lys329. While 228 was reported to exhibit good stability in human hepatocytes (half-life = 20 h) and short half-lives in preclinical pharmacokinetics studies (1 to 2.3 h in the rat and dog), metabolite profiles of the compound from these studies have not been published.(226)
Scheme 34
Scheme 34. Proposed Mechanism of Irreversible Inhibition of GABAAT by 228a
aThe inhibition is initiated by formation of the Schiff base 234 with PLP (a). Arg192 engages the carboxylate moiety to anchor the inhibitor in the active site of the enzyme. Tautomerization (b) of 234 generates the Michael acceptor 236, which is subject to successive hydrolytic reactions (c and d) that liberate 2 equiv of fluoride to ultimately produce 238. Because of conformational restrictions, 236 reacts with H2O rather than the catalytic Lys329 to form an unstable difluorohydrin, which degrades to the acyl fluoride 237 and then to PLP-bound diacid 238. The nascent carboxylate in 238 is positioned to engage in a second electrostatic interaction with Arg445. Hydrolysis of 238 (e) releases PLP-derived amine 239 and (1S)-4-oxocyclopentane-1,3-dicarboxylate (240), which can spontaneously undergo decarboxylation (f) to give (S)-3-oxocyclopentane-1-carboxylate (241).
4. Fluorinated Ethers
ARTICLE SECTIONS
4.1. Fluorinated Alkoxy Substituents
During compound optimization, the substitution of alkoxy groups with fluorinated homologues (e.g., OCH3 to OCF3 or OCHF2) is an approach that has been used to modify both physicochemical properties and metabolite profile.(227,228) As substituents, OCF3 groups usually confer enhanced lipophilicity compared with OCH3. This effect reflects both the higher intrinsic lipophilicity of fluorine (π = 0.14) and the inductive electron-withdrawing effects of fluorine that reduce the hydrogen-bond-accepting (pKHBX) character of the oxygen atom.(227−230) However, exceptions to this trend have been noted, many of which involve a close proximal relationship between a fluorine substituent and an oxygen-containing functionality that is believed to impart increased overall polarity to a molecule, thereby favoring solvation in polar media.(4) Increased lipophilicity is evident in higher measured log D values, but counterintuitively, this substitution may actually detract from passive permeability. Such a trend was noticed during an analysis of matched molecular pairs containing anisole (PhOCH3) and trifluoroanisole (PhOCF3) in the corporate compound collection at Pfizer.(231) The substitution of anisoles with trifluoroalkoxy groups alters the conformational preference of the substituent from coplanar to orthogonal with respect to the aromatic system. This arrangement relieves conformational strain by reducing orbital overlap between the oxygen electron lone pair and the aryl ring. The orthogonal OCF3 substituent is larger and is believed to be more susceptible to steric forces exerted by constituents of the cell membrane that slow its passage across the monolayer, resulting in impaired membrane permeability.
Perhaps the most common reason to explore substitution with OCF3 in place of OCH3 or CH2CH3 is to avert a biotransformation pathway involving O-demethylation and/or to decrease the overall rate of oxidative metabolism.(231−233) This approach is intuitive since the rate-limiting step in an O-demethylation reaction is the abstraction of a hydrogen atom from the CH3O group, a process than cannot occur when the H is replaced by F. However, the success of this approach has been mixed.(228) While reports of reduced metabolic clearance and extended pharmacokinetics of compounds containing OCF3 continue to be described,(229,234) the Pfizer survey found no consistent improvement in metabolic stability for trifluoroanisoles compared to anisoles.(231) Considering the breadth of the data set, the reasons for this lack of a trend are likely varied. In some cases, the OCH3 moiety does not represent the main metabolic soft spot, while in others, the additional lipophilicity conferred by OCF3 may impart stronger affinity for drug-metabolizing enzymes like P450. A third possibility is that metabolizing enzymes may be able to catalyze O-demethylation via an alternative mechanistic route that does not involve the direct cleavage of a C–F bond. Such an explanation was invoked when OSI-930 (242), a c-Kit/VEGFR inhibitor that was advanced into clinical development for the treatment of cancer, was observed ostensibly to undergo O-detrifluoromethylation in a preclinical study using rats and again in a separate experiment using liver microsomes.(235) An ipso substitution pathway that involved the loss of the OCF3 moiety as a leaving group was proposed to explain the observation (Scheme 35). Certain features of this mechanism, including the formation of the hemiketal intermediate 244 and the overall displacement of an alkoxy group by hydroxyl, found precedent in classic work on the bioactivation of phenacetin and acetaminophen that had been performed in the 1970s.(236) In those studies, multiple pathways for the O-deethylation of phenacetin had been described, one of which proceeded with quantitative incorporation of atmospheric 18O at the site of substitution. Hence, it is well-established that metabolic processes that appear to involve O-dealkylation of aromatic ethers may actually proceed by a displacement reaction that involves either ipso substitution or epoxide formation. The extension of this concept to include the displacement of OCF3 groups has been readily accepted since halogens, which are characterized by similar degrees of electronegativity relative to OCF3, are also subject to displacement from aryl rings during metabolism.(237) In the case of OCF3 substituents, the reaction would liberate fluorophosgene (246) as a byproduct of O-dealkylation. Since 246 exists as a gas at standard temperature and pressure, studies of its toxicology have only explored inhalation as the route of administration. For this reason, it is difficult to connect the dose of 246 that might be liberated during the metabolism of a difluoromethoxy-substituted compound to the toxicological effects of direct exposure. However, it is sensible in the interest of safety to take steps to limit the formation of 246 as a metabolite, as its toxicity is known to be severe.(238)
Scheme 35
Scheme 35. Proposed Mechanism of the Metabolism of OSI-930 (242)a
aOSI-930 (242) undergoes single-electron oxidation by P450, creating a radical intermediate that exists in both nitrogen- and carbon-centered resonance forms (243) prior to recombination/rebound hydroxylation. The resulting hemiketal 244 loses CF3OH, which rapidly degrades to fluorophosgene (246) at room temperature, producing the transient quinone imine intermediate 245. Quinone 245 is then reduced to the observed hydroxyphenyl metabolite 247.
The deployment of difluoromethoxy (OCHF2) substituents is intended to strike a balance between the properties of OCH3 and OCF3. The OCHF2 motif has been suggested to offer consistent improvements in several metrics, including lower log D and increased passive cellular permeability relative to OCF3 and slightly lower rates of oxidative metabolism compared with OCH3.(231) Examples of clinically evaluated compounds incorporating metabolically stable OCHF2 substituents are the β-secretase inhibitor 248; the antifibrotic agent FT061 (250), an analogue of the antiallergic drug tranilast (249) that was effective in a rodent model of diabetic nephropathy; and AZD0837 (251), the prodrug of a direct inhibitor of thrombin that was evaluated clinically as a treatment for venous thromboembolism.(239−242) Additional examples include pantoprazole (252), an irreversible proton-pump inhibitor marketed for the treatment of gastroesophageal reflux disease (GERD), CHF 6001 (253), an inhibitor of phosphodiesterase-4 (PDE-4) that was of interest as a potential treatment for pulmonary inflammation, and the long acting PDE-4 inhibitor roflumilast (254) that is approved to treat chronic obstructive pulmonary disease.(243−245) Roflumilast is metabolized to the pyridine N-oxide, an equally active metabolite that is believed to account for 90% of the PDE-4 inhibitory activity of the drug in vivo. The increased electronegativity associated with the OCHF2 substituent of 248 resulted in a reduction in P-glycoprotein-mediated efflux across the blood–brain barrier, enhancing brain penetration for this centrally acting compound.(240) Metabolic stability was also improved relative to compounds with unsubstituted methoxy substituents, presumably because those groups represented soft spots for oxidative metabolism. Furthermore, short-chain alkoxy substituents resulted in improved metabolic stability in liver microsomes relative to longer-chain groups, although the relationship was also influenced by aromatic fluorine substitution at distal sites within the series. This finding reaffirms the point that such trends between structure and metabolic stability are highly context-dependent and may be influenced by remote interactions in a way that can be difficult to anticipate.
The use of OCHF2 moieties to avert O-demethylation has been described in other studies. For example, this structural element is featured in several PDE4 inhibitors that have been explored for their anti-inflammatory effects against aberrant immune cells.(246−249) However, several of these compounds were eventually recognized to carry on-target liabilities that were exacerbated in some cases by high exposure.(245) In light of these issues, intratracheal/intranasal administration of CHF 6001 (253) was explored as a treatment for inflammatory pulmonary disorders, and despite extensive metabolism of the compound in rats prior to disposition, the difluoromethoxy group of [14C]-253 remained intact in all metabolites identified in the urine and feces.(244) Similarly, metabolite profiling studies of AZD0837 (251) and pantoprazole (252) in vivo were completed without any report of biotransformation of the OCHF2 moieties of these compounds.(242,243) Substitution with OCHF2 was explored further during an effort to produce analogues of the antifibrotic agent tranilast (249) with improved pharmaceutical properties.(241) While this initiative was able to produce a candidate (250) with lower clearance in vivo and no associated increase in the volume of distribution, the improvement in pharmacokinetics was ascribed to an increase in the extent of protein binding that helped to sequester the compound from drug-metabolizing enzymes.
Despite these endorsements, the collective experience with OCHF2 groups as a replacement for either OCF3 or OCH3 has not found them to be uniformly inert. Biotransformation studies of BMS-665053 (255), a corticotropin-releasing factor (CRF) receptor-1 antagonist that was investigated as a potential treatment for depression and anxiety, revealed an apparent example of O-dedifluoromethylation as part of a metabolic pathway leading to downstream GSH and glucuronide conjugates in rats.(250) It remains unclear whether the intermediate formed along the bioactivation pathway leading to the GSH conjugate was the epoxide 256 (Scheme 36, path a), which reacted with GSH and expelled formyl fluoride (258)(251) to generate 259, or the quinone imine 261 (Scheme 36, path b), which was formed by direct O-dealkylation by P450 to afford 260 as the precursor. However, the observation of the glucuronide conjugate 262 but not phenol precursor 260 suggested that the latter pathway was at least operative, if not fully responsible for the formation of both metabolites.(250) While the identification of ways to avert bioactivation in this series of CRF receptor antagonists became a focus of the optimization campaign and the evaluation of additional compounds brought the relationship between structure and bioactivation into better focus, the analogues with the best composite profile retained the OCHF2 moiety. However, none of these analogues was able to avoid the formation of reactive metabolites entirely.
Scheme 36
Scheme 36. Proposed Metabolic Pathways to Explain the Formation of Metabolites Observed with the CRF Receptor Antagonist BMS-665053 (255)
The direct displacement of OCHF2 groups by biological thiols has also been demonstrated in the context of the pyrido[2,3-b]pyrazine 263, which was observed to undergo nucleophilic displacement of the 3-substituent by GSH in rat liver S9, a matrix where GSTs are plentiful.(252) Interestingly, the same compound also reacted spontaneously in buffer (pH 7.4, 37 °C) with either of the two peptides Ac-FAACAA or Ac-YACAKASAHA. Positive-ion electrospray MS/MS was used concurrently to show that the OCHF2 group had been displaced by the cysteine residues of these peptides, indicating that the reaction was common among biological thiols and could proceed in the absence of catalytic enzymes (Scheme 37). Thus, while the OCHF2 moiety can confer metabolic stability relative to OCH3 and OCF3 in many circumstances, it can still serve as a leaving group if deployed in the context of heterocycles that are inherently susceptible to nucleophilic displacement reactions.
Scheme 37
Scheme 37. Displacement of the OCHF2 Group in 263 by a Biological Thiol
In contrast to the popularity of the OCHF2 group in drug design, the OCH2F moiety is deployed rarely, and a full understanding of its metabolic pathways is lacking. Perhaps the best known example of a compound exhibiting this functionality is the anesthetic agent sevoflurane (98), which occasionally causes hepatotoxicity.(22) It should be noted that the metabolism of 98 is the result of a complex, multistep pathway (Scheme 18) that results in the eventual cleavage of the OCH2F moiety in the final step of the sequence.(141) Although a few plausible mechanisms have been suggested to explain how this step occurs, evidence to distinguish among them has not been published. Thus, it remains unclear whether the observed loss of OCH2F from 98 has any implications for the metabolism of other compounds incorporating this structural element.
Another motif with low representation in the medicinal chemistry literature is the α,α-difluoroethoxy (OCF2CH3) moiety, which offers interesting properties compared with the OCF3 and CH2CH3 substituents.(227,253) Chromatographic measurements of log P values indicate that the α,α-difluoroethoxy moiety in 270 confers higher polarity than either the OCF3 (268) or CH2CH3 (269) analogue.(254)
However, the OCF2CH3 moiety was recently shown to be hydrolytically sensitive as a function of its chemical environment. Specifically, metabolism of 1-(1,1-difluoroethoxy)-4-methoxybenzene (271) by C. elegans resulted in the unexpected formation of 4-acetoxyphenol (275) instead of the anticipated 4-(1,1-difluoroethoxy)phenol (272).(253) This observation was explained by chemical degradation of the primary phenol metabolite 272 facilitated by improved stability of the oxonium ion 273 compared with the methoxy progenitor 271 (Scheme 38). Notably, α,α-difluoroethyl sulfides, close analogues bearing a sulfur atom in place of oxygen in this series, were not found to be similarly unstable, and metabolism of the sulfur analogue of 271 proceeded via S-oxidation and O-demethylation to afford chemically stable products.
However, the OCF2CH3 moiety was recently shown to be hydrolytically sensitive as a function of its chemical environment. Specifically, metabolism of 1-(1,1-difluoroethoxy)-4-methoxybenzene (271) by C. elegans resulted in the unexpected formation of 4-acetoxyphenol (275) instead of the anticipated 4-(1,1-difluoroethoxy)phenol (272).(253) This observation was explained by chemical degradation of the primary phenol metabolite 272 facilitated by improved stability of the oxonium ion 273 compared with the methoxy progenitor 271 (Scheme 38). Notably, α,α-difluoroethyl sulfides, close analogues bearing a sulfur atom in place of oxygen in this series, were not found to be similarly unstable, and metabolism of the sulfur analogue of 271 proceeded via S-oxidation and O-demethylation to afford chemically stable products.Scheme 38
Scheme 38. Metabolism of 271 by C. elegans, Which Catalyzes O-Demethylation of the Anisole Methyl Moiety to Afford the Hydrolytically Sensitive Product 272
In the two examples reported to date, the 2,2,2-trifluoroethoxy (CF3CH2O) moiety was resistant to metabolism. This finding was based on a comparison of the rates of metabolism of 7-ethoxycoumarin (276) and phenacetin (278), both of which are known substrates of P450 deethylase activity, with those of the 2,2,2-trifluoroethyl analogues 277 and 279.(150) In phenobarbital-induced RLM, the degradation rates were 1.93 and 3.48 nmol min–1 (mg of protein)−1 for 276 and 278, respectively, whereas the rates of metabolism of 277 and 279 were too low to quantify. Additional studies ruled out the possibility that the difference could be ascribed to P450 inhibition by the 2,2,2-trifluoroethoxyl variants. While the origin of the enhanced stability was not explained, the inductive effect of the fluorine substituents is presumably responsible, effectively raising the energy level of the radical intermediate that is produced by H atom abstraction and decreasing the rate of reaction.
4.2. Fluorinated Dioxoles
Geminal difluorination of the benzodioxole moiety is an approach that has attracted attention as a means of improving the pharmaceutical and metabolic properties of drug candidates containing this structural motif, although the literature offers examples of both successful and unsuccessful applications. The 2,2-difluorobenzo[d][1,3]dioxole moiety is often introduced to block metabolism of the methylene analogue, which can inhibit P450 mechanistically by generation of a carbene intermediate that coordinates tightly to the Fe atom.(255) Fludioxonil (280), a nonsystemic fungicide used in agriculture to protect cereal, fruit, and vegetable crops, provides a representative example.(256) Although metabolized extensively following oral administration (100 mg/kg) in rats, with only 12% of the dose recovered as unchanged parent compound in excreta, the difluorodioxole moiety of 280 was intact in all of the metabolites that were identified in those matrices.(256) Similarly, the difluorodioxole moiety of the fatty acid amide hydrolase inhibitor JNJ-42165279 (281) was intact in all of the metabolites characterized preclinically during studies with liver microsomes (mouse, rat, dog, monkey, human) and hepatocytes (rat, dog, monkey, human).(257) These findings were recapitulated during metabolite profiling of plasma samples from rats, dogs, and monkeys following oral administration of the compound. The difluorodioxole moiety was introduced to enhance the metabolic stability of the camptothecin derivative 282, administered as its prodrug 283.(258) Since the difluorobenzodioxole motif tends to resist oxidative metabolism, specifically with respect to the formation of o-quinone metabolites that are prone to redox cycling and nucleophilic attack by biological nucleophiles, it was anticipated that psychedelic empathogens bearing this moiety would offer an improved balance of efficacy and safety. For example, compounds such as MDMA (ecstasy, 284) hold promise in the treatment of certain psychiatric disorders but have been suggested to carry neurotoxic hazards that involve metabolites.(259) Assertions regarding the neurotoxicity of 284 are controversial and have been contradicted by recent clinical work in which the drug was well-tolerated at therapeutic doses.(260) To be sure, such assertions were grounded initially in a high-profile report that was published in 2002 but ultimately retracted once it became clear that the animals in the study had been administered (+)-methamphetamine instead of 284.(261) Unfortunately, the difluoro variant 285 was not active in humans at doses up to 120 mg, a dose that falls near the high end of the dose range of 284 used clinically.(229) This finding serves as a reminder that substituting fluorine for hydrogen can profoundly alter the action of a drug, even as the metabolic properties might be improved.
While lumacaftor (286), a chaperone of the cystic fibrosis transmembrane conductance regulator protein, was excreted mostly intact in humans, small quantities of a catechol metabolite and a downstream sulfate metabolite, which were formed as the result of degradation of the fluorinated benzodioxole ring, were identified.(262) The mechanism of formation of these metabolites was not explained in the regulatory document supplied to the U.S. Food and Drug Administration, and the metabolic fate of 286 has not been elaborated in the published literature. Since the mechanistic route to this metabolite likely differs from that governing the conversion of unsubstituted dioxoles to catechols, in which the rate-limiting step involves abstraction of a hydrogen atom from the dioxole CH2, two alternative pathways that rely on arene oxidation are postulated in Scheme 39. Hypothetically, incorporation of a single equivalent of 18O would be anticipated if this reaction were conducted under controlled conditions using either an atmosphere of 18O2 (A) or 18O-labeled water (B), rendering the two mechanisms easily differentiable. Incidentally, 286 is also a weak competitive inhibitor of CYP2C8 (amodiaquine N-deethylase IC50 = 12 μM) and CYP2C9 (diclofenac 4′-hydroxylase IC50 = 32 μM), contrary to the notion that unsubstituted benzodioxoles inhibit P450 mechanistically via coordination of the metastable carbene intermediate to the Fe atom.(255,263) Thus, there are at least two lines of evidence in addition to chemical reasoning to suggest that an alternative mechanism governing this O,O-dedifluoromethylenation may apply.
Scheme 39
Scheme 39. Two Potential Mechanisms by Which Lumacaftor (286) May Be Metabolized to the Catechol 290a
a(A) Arene oxidation of 286 by P450 affords 287, which degrades via 288. The degradation pathway for 288 could occur by the direct departure of the phenolate with concomitant release of fluorophosgene (246) or via elimination of HF and hydrolysis of the carbonofluoridate product followed by decarboxylation. Collapse to o-quinone 289 is followed by reduction to the catechol 290, which is either excreted or sulfated. (B) Arene oxidation of 286 at an alternate site would afford epoxide intermediate 291, which could degrade hydrolytically to give 292. Rearrangement of this intermediate with loss of H2O and reduction by an overall mechanism analogous to that described above would afford catechol 290 via the o-quinone 289.
An interesting application of difluorinated ethers (ROCF2R′) has been in the design of MBIs of glycosidases, which have attracted attention as therapeutic targets on the basis of their ubiquity and broad range of functions.(200,264,265) In the example depicted in Scheme 40, 293 mimicked the native glycoside and was accepted as a substrate, with the difluoroether removed by the enzyme from the terminal sugar molecule to afford 294 followed by 295.(264) The α,α-difluoroalcohol intermediate 296 readily eliminated fluoride to afford the reactive acyl fluoride 297, which formed a covalent bond that inactivated the glycosidase as the adduct 298. However, the issue with this class of compounds that has prevented their widespread adoption as either drugs or experimental tools has been the tendency of the acyl fluoride 297 to be hydrolyzed by H2O or exit the catalytic pocket of the protein and react with other proteins as unintended collateral targets.
Scheme 40
Scheme 40. Mechanism-Based Glycosidase Inactivation by the Glycoside 293 Exhibiting a Difluorinated Ethera
aThe substituted ether of the glycoside is displaced (294), generating 295 and the difluorinated alcohol 296, which decays quickly to the acyl fluoride 297. The acyl fluoride then reacts covalently with an amino acid residue in the active site, inactivating the enzyme (298), or diffuses away from the enzyme and is either hydrolyzed or captured by an adventitious nucleophile such as a protein.
5. Fluorinated Amines
ARTICLE SECTIONS
5.1. Fluorinated Piperidines
The introduction of fluorine atoms proximal to an amine has found widespread application as a means of reducing basicity or modulating conformation. In turn, these effects can influence pharmacology or interfere with metabolism. While optimization of KSP inhibitors 34–39 offered one example (vide supra), another example was provided by the fentanyl analogue NFEPP (300).(60,266)
In contrast to the more basic 299, the fluorine atom installed in the piperidine ring of 300 restricted protonation to low-pH environments and thereby facilitated selective agonism of μ opioid receptors in damaged peripheral tissues. Similarly, in the phenylethanolamine N-methyltransferase inhibitor 301, the CHF2 substituent represented the optimal compromise compared with the lower and higher fluorinated homologues, conferring selectivity for the target enzyme relative to the α2 adrenoreceptor.(267)
In contrast to the more basic 299, the fluorine atom installed in the piperidine ring of 300 restricted protonation to low-pH environments and thereby facilitated selective agonism of μ opioid receptors in damaged peripheral tissues. Similarly, in the phenylethanolamine N-methyltransferase inhibitor 301, the CHF2 substituent represented the optimal compromise compared with the lower and higher fluorinated homologues, conferring selectivity for the target enzyme relative to the α2 adrenoreceptor.(267)5.2. Fluorinated Pyrrolidines
However, care should be exercised when configuring the relationship between amines and fluorine atoms since some arrangements can be cryptically predisposed toward metabolic activation.(27,268−270) An example is provided by pyrrolidine-based DPP4 inhibitors 302 and 303, 
in which the fluorinated pyrrolidine ring was a source of metabolic activation and covalent binding to protein in RLM.(268) Studies with tritiated compounds revealed that the protein binding was irreversible, dependent on both time and NADPH, and attenuated by the addition of GSH or N-acetylcysteine. By means of incubations conducted in the presence of the hard nucleophile semicarbazide, which captures aldehydes as the imine species, the metabolic pathway depicted in Scheme 41 was elucidated. This bioactivation process was hypothesized to begin with α-hydroxylation of the pyrrolidine ring of 302 or 303 to afford the hemiaminal 304. Ring opening of 304 to give the aldehyde 305 would set the stage for elimination of HF to produce the unsaturated aldehyde 306, a dual electrophile capable of cross-linking proteins. Interception of 306 by GSH would generate 307, which can exist in equilibrium with the hemiaminal 308.(268)
in which the fluorinated pyrrolidine ring was a source of metabolic activation and covalent binding to protein in RLM.(268) Studies with tritiated compounds revealed that the protein binding was irreversible, dependent on both time and NADPH, and attenuated by the addition of GSH or N-acetylcysteine. By means of incubations conducted in the presence of the hard nucleophile semicarbazide, which captures aldehydes as the imine species, the metabolic pathway depicted in Scheme 41 was elucidated. This bioactivation process was hypothesized to begin with α-hydroxylation of the pyrrolidine ring of 302 or 303 to afford the hemiaminal 304. Ring opening of 304 to give the aldehyde 305 would set the stage for elimination of HF to produce the unsaturated aldehyde 306, a dual electrophile capable of cross-linking proteins. Interception of 306 by GSH would generate 307, which can exist in equilibrium with the hemiaminal 308.(268)Scheme 41
Scheme 41. Metabolic Pathway Elucidated for the DPP4 Inhibitors 302 and 303 in RLM
The alternative pathway presented in Scheme 42 was also contemplated. This process relied on β-hydroxylation of the pyrrolidine ring of 302 or 303 to give the fluoroalcohol 309, which collapsed to the epoxide 310 with elimination of HF. Reaction of the epoxide with GSH would then afford a mixture of 311 and 312. However, this mechanism was ruled out on the basis of experiments in which the addition of an epoxide hydrolase inhibitor failed to reduce protein covalent binding. Studies with recombinant rat P450 enzymes indicated that metabolic activation was catalyzed primarily by CYP3A1 and CYP3A2. Consistent with this finding, covalent binding to protein was enhanced using liver microsomes from rats that had been pretreated with pregnenolone-16α-carbonitrile and dexamethasone, compounds that induce the production of these enzymes in vivo.(268)
Scheme 42
Scheme 42. Alternative Epoxide-Based Metabolic Pathway Contemplated for the DPP4 Inhibitors 302 and 303 in RLM
However, the presence of this fluorinated motif is not necessarily a predictor of metabolic activation since studies with several additional 3-fluoropyrrolidine derivatives have been completed without revealing overt susceptibility to such a process.(269−271) Interestingly, all of the examples described are 3,3-difluoro-substituted pyrrolidine derivatives, including the DPP4 inhibitors 313 and 314 and the HIV-1 non-nucleoside reverse transcriptase inhibitor 315. While the half-life of 315 in liver microsomes was short and the difluoropyrrolidine ring was subject to metabolism by a pathway that was not elucidated, there was no clear indication that reactive metabolites were formed. Geminal difluoro substitution would be expected to increase the lipophilicity, which in some cases may enhance the rate of oxidative metabolism by P450 enzymes.
5.3. Fluorinated Azepanes
The metabolic elimination of HF from a γ-difluorinated amine during metabolism studies has been shown to be a pathway involving bioactivation. Detailed profiling of the pan-Pim kinase inhibitor 316
revealed potent time-dependent inhibition (TDI) of CYP3A enzymes with IC50 values of <100 nM.(272) This effect was observed using both testosterone and midazolam as substrates, compounds that bind to different sites of CYP3A, prompting concern about drug–drug interactions. Indeed, with a kinact/Ki ratio of 64, the CYP3A TDI was comparable to that of the reference agent mifepristone. Metabolite identification studies indicated that the fluorinated azepane ring of 316 was a major site of metabolism. Additional studies designed to illuminate the underlying mechanism focused on compounds with simpler substructures in place of the 2-substituted thiazole, seeking to probe aspects of the azepane ring configuration and substitution pattern while exploring the effects of lipophilicity and polarity. These studies, conducted in the context of a benzoyl substituent, established that TDI was not related to the chirality of the amine since the two enantiomers 318 and 319 were similarly problematic (Figure 10).(272) The dimethylamine homologue was free of TDI, but when this modification was installed in the fully elaborated thiazole analogous to 316, CYP3A TDI remained a problem, illustrating a limitation of using truncated substructures as probes. Notably, the simple non-fluorinated amino-substituted azepanes 321 and 322 were free of the liability, an observation that, together with the dimethylamine data, exonerated the nitroso pathway depicted in Scheme 43 (path a) as the source of CYP3A TDI. Since the more lipophilic desamino derivative 320 also demonstrated CYP3A TDI, consideration was given to path c as a possible contributor. However, results of GSH-trapping experiments were consistent with path b, in which α-hydroxylation of the exocyclic amine and loss of NH3 afforded a ketone, setting the stage for elimination of HF to produce the electrophilic α,β-unsaturated homologue. This model was supported by the observation of weak CYP3A TDI with the racemic hydroxyl analogue 323, which could undergo oxidation to give the same ketone intermediate and then follow a similar pathway of chemical activation. Further support came from evaluation of the α-methyl amines 324 and 325, which were unsusceptible to this pathway and confirmed to be free of CYP3A TDI.(272) While the α-methyl derivatives retained pan-Pim kinase inhibitory activity, these compounds were poorly active in cell-based assays. Further optimization of fluorination patterning, which is important as a modulator of membrane permeability and pharmacokinetic properties, resulted in the identification of the β-fluoro derivative GDC-0339 (317) as an early development candidate.(273)
revealed potent time-dependent inhibition (TDI) of CYP3A enzymes with IC50 values of <100 nM.(272) This effect was observed using both testosterone and midazolam as substrates, compounds that bind to different sites of CYP3A, prompting concern about drug–drug interactions. Indeed, with a kinact/Ki ratio of 64, the CYP3A TDI was comparable to that of the reference agent mifepristone. Metabolite identification studies indicated that the fluorinated azepane ring of 316 was a major site of metabolism. Additional studies designed to illuminate the underlying mechanism focused on compounds with simpler substructures in place of the 2-substituted thiazole, seeking to probe aspects of the azepane ring configuration and substitution pattern while exploring the effects of lipophilicity and polarity. These studies, conducted in the context of a benzoyl substituent, established that TDI was not related to the chirality of the amine since the two enantiomers 318 and 319 were similarly problematic (Figure 10).(272) The dimethylamine homologue was free of TDI, but when this modification was installed in the fully elaborated thiazole analogous to 316, CYP3A TDI remained a problem, illustrating a limitation of using truncated substructures as probes. Notably, the simple non-fluorinated amino-substituted azepanes 321 and 322 were free of the liability, an observation that, together with the dimethylamine data, exonerated the nitroso pathway depicted in Scheme 43 (path a) as the source of CYP3A TDI. Since the more lipophilic desamino derivative 320 also demonstrated CYP3A TDI, consideration was given to path c as a possible contributor. However, results of GSH-trapping experiments were consistent with path b, in which α-hydroxylation of the exocyclic amine and loss of NH3 afforded a ketone, setting the stage for elimination of HF to produce the electrophilic α,β-unsaturated homologue. This model was supported by the observation of weak CYP3A TDI with the racemic hydroxyl analogue 323, which could undergo oxidation to give the same ketone intermediate and then follow a similar pathway of chemical activation. Further support came from evaluation of the α-methyl amines 324 and 325, which were unsusceptible to this pathway and confirmed to be free of CYP3A TDI.(272) While the α-methyl derivatives retained pan-Pim kinase inhibitory activity, these compounds were poorly active in cell-based assays. Further optimization of fluorination patterning, which is important as a modulator of membrane permeability and pharmacokinetic properties, resulted in the identification of the β-fluoro derivative GDC-0339 (317) as an early development candidate.(273)Figure 10
Figure 10. Relationships between structure and CYP3A inhibition for a series of azepane derivatives.
Scheme 43
Scheme 43. Potential Metabolic Pathways Giving Rise to CYP3A TDI from Difluorinated Azepane Derivativesa
aProposed metabolic pathways that may result in TDI of P450 by 318: (a) Oxidation of the primary amine in two steps yields the nitroso derivative 326, which coordinates to the heme protein. (b) Oxidative deamination yields the ketone 327, which undergoes elimination to give the α,β-unsaturated carbonyl 328 and then nucleophilic attack by a biological thiol to give 329 and 330. (c) In the des-NH2 series, α-hydroxylation of the azepane ring to give 331 leads to conjugated imine 332 and then 333, which also undergoes nucleophilic attack to give 334.
5.4. Fluorinated Methylcyclopropyl Amines
Unexpected but strongly adverse effects were associated with oral administration of poly-ADP ribose glycohydrolase (PARG) inhibitors incorporating an N-(1-(fluoromethyl)cyclopropyl)sulfonamide substructure, with observations suggesting a role for gut metabolism on the basis of the absence of toxicity following iv dosing.(274) As background, 335 and 336
exhibited short half-lives in vivo that precluded evaluation in animal efficacy models. Subsequent in vitro studies identified the sulfonamide substituent as the major site of metabolism, giving rise to the primary sulfonamide (RSO2NH2). Fluorination of the methyl substituent of the cyclopropylamino group was pursued as an approach to improve the metabolic stability, and in the initial survey, CHF2 and CF3 derivatives were found to be more stable in human liver microsomes (HLM). However, these compounds were 15- and 150-fold weaker enzyme inhibitors, respectively, while the monofluoromethyl analogue was equipotent and more metabolically stable relative to the unsubstituted methyl compound. The limited options for modification at this site focused interest in the monofluoro compounds, which were advanced into pharmacokinetics studies. In these studies, 337 suffered from high clearance, while 338 exhibited lower clearance but was toxic in vivo, producing ataxia, hepatotoxicity, and gastrointestinal bleeding. These issues became a common theme, with 10 of 11 monofluorinated compounds associated with toxicity, while the only outlier, 337, was cleared rapidly. All 10 toxic analogues elicited ataxia, with onset occurring approximately 5 h postdose and with no apparent correlation to plasma exposure, free drug levels, or PARG inhibitory activity. In contrast, a total of 24 non-fluorinated compounds were well-tolerated across doses ranging from 5 to 160 mg/kg. Collectively, these results implicated a common metabolite of the fluorinated derivatives as the ultimate toxicant, although the metabolic pathway was not elucidated.(274)
exhibited short half-lives in vivo that precluded evaluation in animal efficacy models. Subsequent in vitro studies identified the sulfonamide substituent as the major site of metabolism, giving rise to the primary sulfonamide (RSO2NH2). Fluorination of the methyl substituent of the cyclopropylamino group was pursued as an approach to improve the metabolic stability, and in the initial survey, CHF2 and CF3 derivatives were found to be more stable in human liver microsomes (HLM). However, these compounds were 15- and 150-fold weaker enzyme inhibitors, respectively, while the monofluoromethyl analogue was equipotent and more metabolically stable relative to the unsubstituted methyl compound. The limited options for modification at this site focused interest in the monofluoro compounds, which were advanced into pharmacokinetics studies. In these studies, 337 suffered from high clearance, while 338 exhibited lower clearance but was toxic in vivo, producing ataxia, hepatotoxicity, and gastrointestinal bleeding. These issues became a common theme, with 10 of 11 monofluorinated compounds associated with toxicity, while the only outlier, 337, was cleared rapidly. All 10 toxic analogues elicited ataxia, with onset occurring approximately 5 h postdose and with no apparent correlation to plasma exposure, free drug levels, or PARG inhibitory activity. In contrast, a total of 24 non-fluorinated compounds were well-tolerated across doses ranging from 5 to 160 mg/kg. Collectively, these results implicated a common metabolite of the fluorinated derivatives as the ultimate toxicant, although the metabolic pathway was not elucidated.(274)5.5. PLP-Dependent Metabolism of β-Fluorinated Amines
β-Fluorinated amino acids have been designed as MBIs of enzymes where metabolism of the amino acid involves condensation of the primary amine with the aldehyde of PLP to form an imine-based intermediate.(200,275) Enzymes that fall into this class include bacterial alanine racemase and mammalian and nonmammalian amino acid decarboxylases. Inhibition of these enzymes occurs by elimination of fluorine during metabolism, generating an electrophilic intermediate that reacts covalently with the protein.(276−288) As an example, the bacterial alanine racemase inhibitor fludalanine (340), the Cα-deuterated analogue of d-fluoroalanine (339), 
was advanced into clinical trials as a potential anti-infective agent.(277−279) The mechanism of inhibition by 339 entailed deprotonation of the PLP-based intermediate 344 followed by departure of fluoride from 345, as summarized in Scheme 44.(276−279) The loss of fluoride usurped the normal pathway of deprotonation/reprotonation that occurred with the natural substrate alanine to afford 346, an intermediate of low electrophilicity. Exchange of 346 with the catalytic lysine regenerated the PLP-imine precursor 347 and released 2-aminoacrylic acid (348), a nucleophilic enamine that reacted covalently with the cofactor-based imine 347 to produce 349.(276−282) The intermediate 348 could not be intercepted by exogenous thiols, suggesting a rapid reaction with 347. Hydrolysis of 349 was believed to lead to the irreversibly alkylated enzyme 350.(276−282) Both 339 and β-fluoro-l-alanine (351) were processed by the Escherichia coli B alanine racemase with similar partition ratios of approximately 800, although the Km values differed (Table 2).(280−284)
was advanced into clinical trials as a potential anti-infective agent.(277−279) The mechanism of inhibition by 339 entailed deprotonation of the PLP-based intermediate 344 followed by departure of fluoride from 345, as summarized in Scheme 44.(276−279) The loss of fluoride usurped the normal pathway of deprotonation/reprotonation that occurred with the natural substrate alanine to afford 346, an intermediate of low electrophilicity. Exchange of 346 with the catalytic lysine regenerated the PLP-imine precursor 347 and released 2-aminoacrylic acid (348), a nucleophilic enamine that reacted covalently with the cofactor-based imine 347 to produce 349.(276−282) The intermediate 348 could not be intercepted by exogenous thiols, suggesting a rapid reaction with 347. Hydrolysis of 349 was believed to lead to the irreversibly alkylated enzyme 350.(276−282) Both 339 and β-fluoro-l-alanine (351) were processed by the Escherichia coli B alanine racemase with similar partition ratios of approximately 800, although the Km values differed (Table 2).(280−284)Scheme 44
Scheme 44. Mechanism of Inhibition of Bacterial Alanine Racemase by d-Fluoroalanine (339)
Table 2. Kinetic Constants for the Processing of l-Alanine by Escherichia coli B Alanine Racemase and Inhibition by Fluorinated Alanine Derivatives(283,284)
The mechanism of inhibition of alanine racemase by difluoroalanine (341) followed a different pathway (Scheme 45).(280,283,284) Exchange of 341 with the cofactor-bound intermediate 343 produced 352, which collapsed to 353 and then eliminated fluoride to give 354. In contrast to 346, the presence of the fluorine atom in 354 conferred sufficient electrophilicity for this intermediate to react with the catalytic lysine of the enzyme to afford 356, which eliminated the remaining fluorine to give 357.(283,284) Fluoropyruvic acid (355) was released from 354 as a side product by hydrolysis. Hydrolytic decomposition of 357 released PLP (358), leaving alanine derivatized at the catalytic lysine residue (359). However, this intermediate was hydrolytically labile and regenerated the native enzyme with the concomitant release of 2-amino-3-oxopropanoic acid (360), explaining the reversible inhibition of alanine racemase by 341. Both the d and l isomers of 341 were processed by the Escherichia coli B alanine racemase with similar Km values (116 and 102 mM, respectively), but the partition ratios were higher than for the monofluoro derivative 339 (Table 2).(283,284)
Scheme 45
Scheme 45. Mechanism of Inhibition of Bacterial Alanine Racemase by Difluoroalanine (341)
The presence of the third fluorine atom in the racemate of trifluoroalanine (342) conferred efficient inhibition of Escherichia coli B alanine racemase, with a partition ratio of 10, although the Vmax was low at <0.1 min–1.(283,284) Transamidation of 342 with 343 afforded 361, which tautomerized and lost fluoride to form the intermediate 362 (Scheme 46).(283,284) The difluorinated alkene of 362 was more electrophilic than 354 and reacted with the catalytic lysine with elimination of a second fluorine atom. This step was followed by hydrolysis, with loss of the final fluorine from 342, to give 363. This intermediate readily decarboxylated and released PLP hydrolytically to afford 364. This step left the catalytic lysine acylated by a glycine moiety and inactivated the enzyme irreversibly.(283,284)
Scheme 46
Scheme 46. Mechanism of Inhibition of Bacterial Alanine Racemase by Trifluoroalanine (342)
Fluorovinylglycine (365) followed a trifurcated pathway during processing by Escherichia coli B alanine racemase (Scheme 47).(276,277,285,286) Exchange of 365 with the enzyme lysine in the cofactor-bound 343 afforded 366, which collapsed to 367. This molecule followed the normal enzymatic pathway to lose fluoride to produce the reactive allene 368. The majority (57%) of 368 suffered hydrolytic decomposition to afford electrophilic 2-oxobut-3-enoic acid (374). However, approximately 12% of 368 reacted with the catalytic lysine to provide 369 and then 370, which was hydrolyzed to regenerate the active enzyme. Hydrolysis of 371 afforded 2-amino-3-oxobutanoic acid (372), which underwent decarboxylation to afford aminoacetone (373). The remainder (31%) of 368 reacted with a proximal noncatalytic lysine to afford the adduct 375, a Michael acceptor that reacted with the phenolic hydroxyl of a nearby tyrosine residue to generate 376. This unstable aminal intermediate degraded to produce the irreversibly inactivated enzyme 377 that retained an alkylated tyrosine residue.(276,277,285,286)
Scheme 47
Scheme 47. Mechanism of Inhibition of Bacterial Alanine Racemase by Fluorovinylglycine (365)
The phosphonic acid derivatives 378–380, analogues of 339, 341, and 342, respectively, are also time-dependent inhibitors of Pseudomonas sp. ATCC 23,715 alanine racemase.(289) Compound 378 was the most potent of these derivatives, although detailed mechanistic studies of this inhibitor have not been performed.
5.5.1. Metabolism of β-Fluorinated Amines by Amino Acid Decarboxylases
β-Fluoroamino acid derivatives have also been evaluated as MBIs of the amino acid decarboxylases that are involved in the biosynthesis of aminergic neurotransmitters.(290−297) The quaternary amino acid derivatives 381, 382, and 383 are MBIs of glutamate, histidine, and dopa decarboxylases, respectively, while 384 and eflornithine (385) are inhibitors of ornithine decarboxylase.(290−297)
These fluorinated analogues of the natural substrates are accepted by the decarboxylase enzymes as substrates and converted to their PLP-based imines 387, which are metabolized along the natural pathway (Scheme 48). However, the relative structural arrangement between the fluorine and the acid moiety is such that decarboxylation of 387 leads to the loss of fluoride from 388, producing the electrophilic intermediate 389. This electrophile reacts with the enzyme to produce the covalent intermediate 390. Hydrolysis of 390 ultimately captures the protein as the alkylated derivative 392, while PLP is converted to the amine 239.(290−293) The difluoromethylated derivative of ornithine, 385, is an MBI of ornithine decarboxylase that has been used clinically as an anticancer agent. Inhibition of ornithine decarboxylase by 385 prevented the biosynthesis of the downstream polyamine metabolites spermine and spermidine, which are regulators of cell growth.(294−299) Interestingly, the l derivative of 385 inhibited ornithine decarboxylase 10-fold more potently than the d isomer, although the PLP derivatives of both enantiomers were substrates of the enzyme. This outcome is surprising since the enzyme processes the l isomer of ornithine 10 000 times faster than the d isomer.(294−297) An infusion formulation of 385 has been used as part of a drug combination to treat the second stage of African trypanosomiasis (sleeping sickness) on the basis of inhibition of the ornithine decarboxylase of Trypanosoma brucei gambiense.(298−300) In addition, 385 is marketed as a topical treatment for facial hirsutism in women under the trade name Vaniqa.(300)
These fluorinated analogues of the natural substrates are accepted by the decarboxylase enzymes as substrates and converted to their PLP-based imines 387, which are metabolized along the natural pathway (Scheme 48). However, the relative structural arrangement between the fluorine and the acid moiety is such that decarboxylation of 387 leads to the loss of fluoride from 388, producing the electrophilic intermediate 389. This electrophile reacts with the enzyme to produce the covalent intermediate 390. Hydrolysis of 390 ultimately captures the protein as the alkylated derivative 392, while PLP is converted to the amine 239.(290−293) The difluoromethylated derivative of ornithine, 385, is an MBI of ornithine decarboxylase that has been used clinically as an anticancer agent. Inhibition of ornithine decarboxylase by 385 prevented the biosynthesis of the downstream polyamine metabolites spermine and spermidine, which are regulators of cell growth.(294−299) Interestingly, the l derivative of 385 inhibited ornithine decarboxylase 10-fold more potently than the d isomer, although the PLP derivatives of both enantiomers were substrates of the enzyme. This outcome is surprising since the enzyme processes the l isomer of ornithine 10 000 times faster than the d isomer.(294−297) An infusion formulation of 385 has been used as part of a drug combination to treat the second stage of African trypanosomiasis (sleeping sickness) on the basis of inhibition of the ornithine decarboxylase of Trypanosoma brucei gambiense.(298−300) In addition, 385 is marketed as a topical treatment for facial hirsutism in women under the trade name Vaniqa.(300)Scheme 48
Scheme 48. Mode of Inhibition of Amino Acid Decarboxylases by β-Fluorinated Amino Acid Derivatives 386
PLP-dependent decarboxylases are also susceptible to α-(1′-halo)vinyl-α-amino acid MBIs.(301,302) One example is d-(1-chlorovinyl)glycine (393), which inhibited the alanine racemase from Escherichia coli B.(285,303)
Similarly, (1-fluorovinyl)glycine (394), an MBI of Escherichia coli B alanine racemase, also inhibited Salmonella typhimurium tryptophan synthase. Compound 395 is an inhibitor of lysine decarboxylase from the Gram-negative bacterium Hafnai alvei.(301) The proposed mode of inhibition of S. typhimurium tryptophan synthase by 394 is outlined in Scheme 49. The elimination of HF from the PLP-bound intermediate 397 was believed to give rise to the electrophilic allene 398, which reacted with a lysine residue in the enzyme to afford 399. Sequential tautomerization to 400 and 401 followed by decarboxylation afforded 402, which underwent hydrolysis and liberation of aminoacetone (373), a step that released PLP and restored enzyme function. The mode of action of the lysine decarboxylase inhibitor 395, which rapidly inactivated the enzyme with a Ki = 630 μM and a half-life of 2.8 min, has not been characterized. However, while 395 shares the natural configuration of lysine, the enantiomer 396 performed similarly as a covalent inhibitor, with a Ki of 470 μM and an inactivation half-life of 3.6 min.(301)
Similarly, (1-fluorovinyl)glycine (394), an MBI of Escherichia coli B alanine racemase, also inhibited Salmonella typhimurium tryptophan synthase. Compound 395 is an inhibitor of lysine decarboxylase from the Gram-negative bacterium Hafnai alvei.(301) The proposed mode of inhibition of S. typhimurium tryptophan synthase by 394 is outlined in Scheme 49. The elimination of HF from the PLP-bound intermediate 397 was believed to give rise to the electrophilic allene 398, which reacted with a lysine residue in the enzyme to afford 399. Sequential tautomerization to 400 and 401 followed by decarboxylation afforded 402, which underwent hydrolysis and liberation of aminoacetone (373), a step that released PLP and restored enzyme function. The mode of action of the lysine decarboxylase inhibitor 395, which rapidly inactivated the enzyme with a Ki = 630 μM and a half-life of 2.8 min, has not been characterized. However, while 395 shares the natural configuration of lysine, the enantiomer 396 performed similarly as a covalent inhibitor, with a Ki of 470 μM and an inactivation half-life of 3.6 min.(301)Scheme 49
Scheme 49. Mode of Inhibition of Tryptophan Synthase by 394
In addition to the fluorinated alkenes that were investigated as inhibitors of GABAAT (vide supra), fluorinated amino acids have also been studied. 4-Amino-3-fluorobutanoic acid (404, F-GABA) 
was evaluated as a probe for GABAAT and found to be a substrate but not an inhibitor of the enzyme.(222,304,305) The presence of the fluorine atom in 404 diverted the metabolic pathway away from the normal process of transamination in favor of enzymatic loss of HF that was dependent on the absolute configuration of the C–F center. Evaluation of the resolved enantiomers revealed different behaviors, with (R)-404 eliminating HF 10-fold faster and with 20-fold greater efficiency than the antipodal (S)-404. In addition, (R)-404 was a competitive inhibitor of GABAAT (Km = 49 μM), whereas (S)-404 did not inhibit the enzyme at a concentration of 1 mM. These observations were attributed to the mode of HF elimination. Experimental and theoretical studies of fluoride elimination indicated that a nonconcerted E1cb mechanism, in which the electron-deficient fluorine atom stabilized the β-anion, was preferred over a concerted E2 mechanism. On the basis of the geometries illustrated in Scheme 50a, (R)-404 could only eliminate via an E1cb mechanism because of the syn relationship between the γ-S proton in the PLP adduct (R)-405.(305,306) This elimination reaction gave (R)-406 as an intermediate. Exchange of the PLP of 407 with GABAAT released (E)-4-aminobut-3-enoic acid (408), which hydrolyzed to succinic semialdehyde (409). The other isomer, (S)-404, characterized by a trans relationship between the γ-S proton and fluorine, could avail of either an E1cb or an E2 mechanism, with the latter depicted in Scheme 50b. The profiles of (R)-404 and (S)-404 as substrates suggested that the GABAAT enzyme recognized the stereochemical relationship between the carboxylate and ammonium moieties depicted in Figure 11a. Isomer (R)-404 is a low-energy conformer because of the favorable gauche relationship between the NH3+ group and F atom. The analogous conformation for (S)-404 (Figure 11c), in which the F atom is anti to the protonated amine, has a higher energy than those depicted in Figure 11b,d.(306) Thus, because (R)-404 adopted the conformation in solution that was recognized initially by the enzyme, it was accepted more readily as a substrate. Collectively, these observations led to the conclusion that the E1cb mechanism of HF elimination predominated in both cases, with (S)-404 eliminating more slowly because of the Km rather than the kcat or Vmax parameters.(304−307)
was evaluated as a probe for GABAAT and found to be a substrate but not an inhibitor of the enzyme.(222,304,305) The presence of the fluorine atom in 404 diverted the metabolic pathway away from the normal process of transamination in favor of enzymatic loss of HF that was dependent on the absolute configuration of the C–F center. Evaluation of the resolved enantiomers revealed different behaviors, with (R)-404 eliminating HF 10-fold faster and with 20-fold greater efficiency than the antipodal (S)-404. In addition, (R)-404 was a competitive inhibitor of GABAAT (Km = 49 μM), whereas (S)-404 did not inhibit the enzyme at a concentration of 1 mM. These observations were attributed to the mode of HF elimination. Experimental and theoretical studies of fluoride elimination indicated that a nonconcerted E1cb mechanism, in which the electron-deficient fluorine atom stabilized the β-anion, was preferred over a concerted E2 mechanism. On the basis of the geometries illustrated in Scheme 50a, (R)-404 could only eliminate via an E1cb mechanism because of the syn relationship between the γ-S proton in the PLP adduct (R)-405.(305,306) This elimination reaction gave (R)-406 as an intermediate. Exchange of the PLP of 407 with GABAAT released (E)-4-aminobut-3-enoic acid (408), which hydrolyzed to succinic semialdehyde (409). The other isomer, (S)-404, characterized by a trans relationship between the γ-S proton and fluorine, could avail of either an E1cb or an E2 mechanism, with the latter depicted in Scheme 50b. The profiles of (R)-404 and (S)-404 as substrates suggested that the GABAAT enzyme recognized the stereochemical relationship between the carboxylate and ammonium moieties depicted in Figure 11a. Isomer (R)-404 is a low-energy conformer because of the favorable gauche relationship between the NH3+ group and F atom. The analogous conformation for (S)-404 (Figure 11c), in which the F atom is anti to the protonated amine, has a higher energy than those depicted in Figure 11b,d.(306) Thus, because (R)-404 adopted the conformation in solution that was recognized initially by the enzyme, it was accepted more readily as a substrate. Collectively, these observations led to the conclusion that the E1cb mechanism of HF elimination predominated in both cases, with (S)-404 eliminating more slowly because of the Km rather than the kcat or Vmax parameters.(304−307)Scheme 50
Scheme 50. (a) E1cb Mechanism of HF Elimination from (R)-404; (b) E2 Mechanism of HF Elimination from (S)-404
Figure 11
Figure 11. Conformations of (a) (R)-404 and (c) (S)-404 recognized initially by GABAAT and (b, c) the two low-energy conformations of (S)-404.
Lesogaberan (410), a GABAB agonist that was developed for the treatment of gastroesophageal reflux disease, also appears to be a substrate for GABAAT.(308,309) A metabolite profiling study in rats revealed that 410 was converted to multiple metabolites, including two that were devoid of the fluorine atom: (2-carboxyethyl)phosphinate (416) and (3-hydroxypropyl)phosphinate (417).(309) The former was the most abundant of all of the metabolites detected in urine. While the intended target of 410 was located in the periphery, autoradiography following administration of radiolabeled drug to rats revealed distribution into certain CNS structures, including the area postrema, median eminence, and pineal gland, a pattern that was reminiscent of the distribution of GABA itself.(310) This distribution pattern, together with similarities between the defluorinated urinary metabolites of 410 and those of 404, prompted the suggestion that 410 might be metabolized in the CNS. The proposed metabolic pathway for 410 is illustrated in Scheme 51. The first step is the formation of the Schiff base 411 by exchange with PLP-bound GABAAT. By analogy to the mechanism illustrated in Scheme 50, this intermediate would suffer defluorination (412) to provide the unsaturated imine 413, which would exchange with the lysine of GABAAT to release PLP and the enamine 414. Hydrolysis of 414 would afford aldehyde 415, which, although not observed directly, presumably would leave the CNS for the periphery, where it would be either oxidized to 416 or reduced to 417, the two defluorinated products that were excreted. It should be emphasized that the involvement of GABAAT in the metabolism of 410 has not been shown directly but is plausible on the basis of the similarity to the known GABAAT substrate 404 and the similarities between the tissue distributions of 410 and GABA.(310)
Scheme 51
Scheme 51. Proposed Mechanism of Defluorination of 410 by GABAAT and Further Metabolism to Give 416 and 417
Fludalanine (340) features a deuterium label that was introduced to improve the metabolic stability, and it carries the distinction of being the first such drug candidate that was advanced into clinical trials.(311) In this case, the enzyme involved in the metabolism of 340 was d-amino acid oxidase (DAAO), which catalyzes oxidative deamination to afford the toxic metabolite fluoropyruvic acid (355) (Scheme 52). The introduction of deuterium in 340 reduced the rate of oxidation 2- to 3-fold relative to the protio analogue 339. Pyruvate dehydrogenase, a thiamine diphosphate (419)-dependent enzyme, accepted 355 as a substrate and processed it initially in analogy with pyruvic acid (420). This compound was successively transformed via 421–425 to the CoA ester of acetic acid (426), as depicted in Scheme 53, path a. However, the presence of fluorine in 355 led to decarboxylative defluorination of the adduct 427 to produce 428, the tautomer of 422, which could react with nucleophiles (Scheme 53, path b).(312) Alternatively, processing of 355 by lactate dehydrogenase formed fluorolactic acid (418). While toxicity associated with fluoride release by pyruvate dehydrogenase was observed in acute studies, long-term toxicology studies in the rat and rhesus monkey revealed that exposure to 418 resulted in intramyelinic vacuolation in the corpus callosum of the brain.(313) The measured levels of 418 in normal healthy volunteers afforded a therapeutic margin of approximately 10-fold. However, in patients with chronic obstructive pulmonary disease the levels of 418 were 2-fold higher, prompting the termination of 340 in 1984.
Scheme 52
Scheme 52. Metabolic Fate of 340
Scheme 53
Scheme 53. Pyruvate Dehydrogenase Uses the Cofactor Thiamine Diphosphate (419) to Effect the Decarboxylative Degradation of 355
6. α-Fluorinated Carbonyl Derivatives
ARTICLE SECTIONS
The metabolism of the mono-α-fluoroketones 40 and 119 was described earlier (Schemes 7 and 19, respectively). The former was metabolized to fluoroacetic acid (5), while the latter appeared to undergo defluorination to afford the ketone.(54,56,61,148) In another interesting example, the Ca2+-dependent large-conductance potassium (Maxi-K) channel opener flindokalner (430, BMS-204352, MaxiPost) was explored as a treatment for the sequelae of a stroke.(314,315) The metabolism of 430 illustrated the potential of an α-fluorinated carbonyl derivative to liberate fluoride metabolically. Initial indications of unusual behavior on the part of this drug were encountered during a disposition study of [14C]-430 administered intravenously to rats, where a majority of the radioactivity in the plasma could not be extracted by organic solvents.(314) Analysis of plasma proteins using gel electrophoresis demonstrated that the administered radioactivity remained associated with specific proteins, particularly albumin, following the separation. Acid-catalyzed digestion of these plasma proteins following administration of a larger dose of [14C]-430 resulted in the liberation of the lysine adduct 434 derived from desfluoro des-O-methyl [14C]-430. The bioactivation sequence proposed to explain the observation involved O-demethylation of the methoxyphenyl moiety of 430 by P450 to afford the phenol 431 as the triggering event (Scheme 54).(314) The formation of 431 set the stage for the elimination of HF to give the electrophilic o-quinone methide 432, which reacted with a Lys residue in serum albumin to give 433. Digestion of a plasma protein sample following exposure to 430 led to the isolation of the lysine adduct 434, in which the side-chain amine was bound to the same carbon atom of 430 that had been substituted with fluorine. These results suggested that 432 has sufficient chemical stability to leave the liver and reenter circulation to react with albumin.(314)
Scheme 54
Scheme 54. Proposed Bioactivation of 430 to Give Electrophile 432 and Trapping by a Lys of Serum Albumin
Whereas the structural and electronic relationship between the anisole moiety and the α-fluorinated carbonyl group set the stage for the bioactivation of 430 and the facile elimination of HF, no such activation of the α-fluorinated carbonyl motif of PF-06650833 (435) would be anticipated. The latter compound is a contemporary example of an inhibitor of interleukin-1 receptor associated kinase 4 (IRAK4) that has entered clinical development for the treatment of autoimmune diseases.(316) While 435 is reported to possess an attractive composite profile, information on the metabolic fate, reactivity, or specific benefit conferred by the α-fluorocarbonyl moiety has not been disclosed.
7. Fluorinated Aromatics
ARTICLE SECTIONS
7.1. Fluorinated Aromatics That Undergo Oxidative Defluorination or Bioactivation
Fluorine substitution is an approach that has been used commonly in medicinal chemistry to interfere with metabolic activation of aromatic rings. Under normal circumstances, oxidative metabolism of an aromatic ring may lead to the production of an arene oxide that is susceptible to nucleophilic attack by a biological nucleophile such as GSH.(1,2,21−23) The electron-withdrawing properties of fluorine can reduce the electron density of the aromatic ring and lower the rate of oxidation by P450, although the net effect of fluorination on the metabolic stability can be modest.(317,318) An example is provided by 437, the 2,10-difluoro-substituted analogue of the anticonvulsant agent carbamazepine (436), which is widely used to treat epilepsy and trigeminal neuralgia.(319)
The clinical hepatotoxicity of 436 is well-documented, with several hundred cases reported in the literature. These cases appear to involve hypersensitivity or an immunological response to a metabolically generated drug–protein complex, implicating oxidative metabolism. In HLM, both arene oxidation and 10,11-epoxidation of 436 have been characterized, the latter producing a pharmacologically active but weakly electrophilic metabolite. In rat hepatocytes, 437 was not subject to oxidative dehalogenation or GSH conjugation, and while some formation of the 10,11-epoxide occurred, aromatic hydroxylation was not observed. The 2-fluoro analogue was subject to monohydroxylation as a minor pathway, and complete inhibition of aromatic hydroxylation required either difluorination (437) or monochlorination of 436.(319)
The clinical hepatotoxicity of 436 is well-documented, with several hundred cases reported in the literature. These cases appear to involve hypersensitivity or an immunological response to a metabolically generated drug–protein complex, implicating oxidative metabolism. In HLM, both arene oxidation and 10,11-epoxidation of 436 have been characterized, the latter producing a pharmacologically active but weakly electrophilic metabolite. In rat hepatocytes, 437 was not subject to oxidative dehalogenation or GSH conjugation, and while some formation of the 10,11-epoxide occurred, aromatic hydroxylation was not observed. The 2-fluoro analogue was subject to monohydroxylation as a minor pathway, and complete inhibition of aromatic hydroxylation required either difluorination (437) or monochlorination of 436.(319)However, fluorine is a modest electron-withdrawing substituent (σp = 0.06; σm = 0.34), and aryl fluorides can be substrates for oxidative metabolism.(317,320,321) The applicable pathways include oxidation with the retention of fluorine to afford fluorinated phenols, oxidation with the loss of fluorine to form phenols, or the formation of GSH conjugates. Aromatic hydroxylation of the series of fluorobenzenes 438–442
by P450 was investigated in Wistar rats using 19F NMR spectroscopy, providing mechanistic evidence of a fluorine NIH shift.(322) The results demonstrated that fluorobenzenes 443 were converted in vivo to phenolic metabolites 447, in which the F atom had rearranged via an NIH-type shift (Scheme 55). However, these metabolites were minor and were overshadowed by products of ring hydroxylation that formed without loss or rearrangement of fluorine. Taken at face value, these results would suggest that oxidation accompanied by a fluorine NIH shift contributed little to the metabolism of fluorobenzenes in vivo. However, considering the reactive nature of the presumed epoxide intermediate (439), some of this material may have been lost to an alternative metabolic pathway. The latter possibility was supported by incubations in liver microsomes that showed the formation of a substantial amount of an NIH-shifted product during the metabolism of 1,4-difluorobenzene (438) in vitro.(322) The addition of GSH and liver cytosol to microsomal incubations lowered the relative abundance of the NIH-shifted phenolic metabolites by 26%, indicating GSH conjugation as a possible competing pathway. Thus, the low yield of fluorine NIH-shifted products during the metabolism of fluoro-substituted benzene derivatives in vivo can, at least in part, be ascribed to alternative pathways for metabolism of the epoxide. GSH conjugation is one potential alternative pathway, facilitated by the enhanced reactivity of fluorinated arene oxides. These fluorinated arene oxides exhibit lower calculated ELUMO values than those substituted by chlorine, implying faster reactions between fluorine-substituted arene oxides and nucleophiles.(322)
by P450 was investigated in Wistar rats using 19F NMR spectroscopy, providing mechanistic evidence of a fluorine NIH shift.(322) The results demonstrated that fluorobenzenes 443 were converted in vivo to phenolic metabolites 447, in which the F atom had rearranged via an NIH-type shift (Scheme 55). However, these metabolites were minor and were overshadowed by products of ring hydroxylation that formed without loss or rearrangement of fluorine. Taken at face value, these results would suggest that oxidation accompanied by a fluorine NIH shift contributed little to the metabolism of fluorobenzenes in vivo. However, considering the reactive nature of the presumed epoxide intermediate (439), some of this material may have been lost to an alternative metabolic pathway. The latter possibility was supported by incubations in liver microsomes that showed the formation of a substantial amount of an NIH-shifted product during the metabolism of 1,4-difluorobenzene (438) in vitro.(322) The addition of GSH and liver cytosol to microsomal incubations lowered the relative abundance of the NIH-shifted phenolic metabolites by 26%, indicating GSH conjugation as a possible competing pathway. Thus, the low yield of fluorine NIH-shifted products during the metabolism of fluoro-substituted benzene derivatives in vivo can, at least in part, be ascribed to alternative pathways for metabolism of the epoxide. GSH conjugation is one potential alternative pathway, facilitated by the enhanced reactivity of fluorinated arene oxides. These fluorinated arene oxides exhibit lower calculated ELUMO values than those substituted by chlorine, implying faster reactions between fluorine-substituted arene oxides and nucleophiles.(322)Scheme 55
Scheme 55. Aromatic Oxidation Accompanied by the NIH Shift of Fluorine in Fluorobenzenes
In a metabolism study that exemplified a fluorine NIH shift, urinary metabolites were profiled following oral administration of the quinoxaline-based HIV-1 non-nucleoside reverse transcriptase inhibitor GW420867X (448) to rabbits, mice, and humans.(323) As captured in Scheme 56, 448 was metabolized extensively on the fluorophenyl ring to form hydroxylated metabolites and glucuronide conjugates in all species. The phenolic metabolites and corresponding glucuronide conjugates were isolated by semipreparative HPLC, and the relative proportions of fluorine-containing metabolites were determined by 19F NMR analysis. The NMR spectra of the isolated metabolites 449 and 450 indicated that the fluorine atom on the phenyl ring had undergone an NIH shift, whereas the composite analytical data of a third metabolite 451 implicated oxidative defluorination as a competing pathway that could originate from the epoxide precursors of 449 and 450. The characterization of the NIH-shifted metabolites in urine from the preclinical species helped facilitate the subsequent identification of these metabolites in human plasma in the clinical trials.(323)
Scheme 56
Scheme 56. Metabolism of 448, a Quinoxaline-Based Non-Nucleoside Inhibitor of HIV-1 Reverse Transcriptase
The fluorination pattern of the hedgehog pathway inhibitor 452 was designed to reduce the electron density of the benzyl moiety and thereby lower its susceptibility to oxidative metabolism.(324) However, metabolite profiling studies with 452 in HLM identified a metabolite, proposed to be the quinone methide 454, that formed during oxidative defluorination of the benzyl moiety. Quinone methide 454 was sufficiently stable to be separated and characterized on a small scale by LC–MS, but this technique did not allow the quinone methide regiochemistry and olefin geometry to be determined (Scheme 57).(324) In reaction-phenotyping studies, metabolic activation of 452 was NADPH-dependent and catalyzed primarily by CYP3A4. One mechanism of bioactivation that was proposed relied on epoxidation of the fluorophenyl ring to afford 453 (Scheme 57). This arene oxide would rearrange to 454, a compound that is stabilized by resonance delocalization of the lone pair of electrons on the piperazine N atom. An alternative proposal envisioned H atom abstraction by P450 from the benzylic site and subsequent oxidation of the delocalized radical.(324) In addition to the quinone methide 454, a GSH adduct of undefined regiochemistry, 455, was identified along with a hydroxylated analogue of this metabolite.(325) Consideration was also given to the potential for the GSH adduct 455 to arise by direct reaction with the epoxide intermediate 453.
Scheme 57
Scheme 57. Potential Metabolic Activation Pathways of the Hedgehog Pathway Inhibitor 452
Tegobuvir (456, TGV) is a mechanistically interesting inhibitor of HCV RNA replication that was discovered through a phenotypic screening approach assessing subgenomic viral replication in replicons.(326) Although this compound was advanced into clinical trials, where it demonstrated antiviral activity in patients infected with genotype 1 chronic HCV infection, its mode of action was enigmatic. While the resistance mutations that arose in response to exposure to 456 in the replicon system could be mapped to the NS5B polymerase protein, the compound failed to inhibit NS5B enzymatic activity in biochemical assays, a conundrum that suggested a more complex antiviral mechanism. Studies to elucidate the mechanism of action revealed that treatment of HCV subgenomic replicon cells with 456 resulted in a modified form of the NS5B protein that exhibited altered mobility on a Western blot SDS-PAGE gel. The modified protein was characterized further as an adduct carrying an additional mass of 820 Da. Formation of the adduct protein did not require the presence of other HCV proteins but was dependent on both functional CYP1A1 enzyme activity and the presence of cellular GSH. Further insight into the inactivation mechanism was provided by the results of mass spectrometric analysis of NS5B that had been purified from a heterologous expression system and treated with 456.(326) The NS5B protein adduct afforded by this process (462 and/or 463) was characterized as a GSH conjugate of defluorinated 456 that had reacted with a Cys residue of the enzyme. On the basis of the experimental observations, a metabolic pathway was proposed involving epoxidation of the fluorobenzene ring of 456 as the triggering event (Scheme 58). Ring opening of the epoxide of 457, facilitated by delocalization of the pyridine N atom electron lone pair, would afford an intermediate that could degrade by the loss of fluoride. This process would produce the highly electrophilic ketone 458, which has multiple potential sites for adduction with GSH. GSH addition by pathways a and b followed by tautomerization would afford the phenolic adducts 459 and 460. However, GSH addition by pathway c would result in the production of the cyclic dienone 461, which could tautomerize to a phenol. This dienone would retain two exposed electrophilic centers capable of reacting with a Cys residue of HCV NS5B, with 462 and/or 463 representing the observed protein adduct.(326) While the specific residue of HCV NS5B that was decorated by 461 was not identified, Cys366, which is proximal to the active site of the enzyme and has been shown to be susceptible to reaction with covalently acting HCV NS5B inhibitors, was a plausible candidate.(327)
Scheme 58
Scheme 58. Proposed Mode of Inhibition of HCV NS5B Polymerase by 456 in HCV Replicons
The BACE-1 inhibitor 464, which incorporates a 5-fluoro-2-aminopyrimidine heterocycle, 
was identified as a promising lead on the basis of its performance in cell-based assays but was associated with CYP3A4 TDI that was traced to metabolic activation of the fluorinated heterocycle.(328) Studies revealed that the bioactivation of this moiety resulted in an oxidative defluorination to give the GSH conjugate 467 and the pyrimidin-5-ol 468, a metabolic pathway that was responsible for the observed TDI (Scheme 59). The proposed metabolic pathway relied on oxidation of the fluoropyrimidine ring to afford an epoxide intermediate followed by reaction with GSH to give an unstable fluorohydrin adduct. This adduct would lose HF to afford 466, which would then tautomerize to the observed product 467. Alternatively, epoxide ring opening with loss of fluoride, as the result of anchimeric assistance by the pyrrolidine N atom, would produce a highly electrophilic quinone iminium derivative that could either add GSH to produce 467 or be reduced to give 468.(328) Further support for this postulate was provided by studies of the fluorophenyl and unsubstituted pyrimidine analogues, neither of which resulted in CYP3A4 TDI (IC50 > 30 μM). Additional structure–liability studies confirmed that dual substitution at the 4- and 6-positions of the 5-fluoropyrimidine ring was sufficient to ameliorate the TDI. This protective effect was exemplified by the 4,6-dimethyl-5-fluoropyrimidine analogue 465, which demonstrated 10-fold weaker inhibition of CYP3A4 relative to 464.(328)
was identified as a promising lead on the basis of its performance in cell-based assays but was associated with CYP3A4 TDI that was traced to metabolic activation of the fluorinated heterocycle.(328) Studies revealed that the bioactivation of this moiety resulted in an oxidative defluorination to give the GSH conjugate 467 and the pyrimidin-5-ol 468, a metabolic pathway that was responsible for the observed TDI (Scheme 59). The proposed metabolic pathway relied on oxidation of the fluoropyrimidine ring to afford an epoxide intermediate followed by reaction with GSH to give an unstable fluorohydrin adduct. This adduct would lose HF to afford 466, which would then tautomerize to the observed product 467. Alternatively, epoxide ring opening with loss of fluoride, as the result of anchimeric assistance by the pyrrolidine N atom, would produce a highly electrophilic quinone iminium derivative that could either add GSH to produce 467 or be reduced to give 468.(328) Further support for this postulate was provided by studies of the fluorophenyl and unsubstituted pyrimidine analogues, neither of which resulted in CYP3A4 TDI (IC50 > 30 μM). Additional structure–liability studies confirmed that dual substitution at the 4- and 6-positions of the 5-fluoropyrimidine ring was sufficient to ameliorate the TDI. This protective effect was exemplified by the 4,6-dimethyl-5-fluoropyrimidine analogue 465, which demonstrated 10-fold weaker inhibition of CYP3A4 relative to 464.(328)Scheme 59
Scheme 59. Proposed Bioactivation Pathway for the BACE-1 Inhibitor 464, a 5-Fluorinated Pyrimidine Derivative Associated with CYP3A4 TDI
Other fluorinated drugs that release fluoride in vivo include the anesthetic agent sevoflurane (98) (vide supra)(132,334) and voriconazole (469), an antifungal agent that became the subject of safety concerns following the observation of periostitis in a heart transplant patient receiving long-term therapy.(329) A group of 10 post-transplant subjects who received 469 at an oral dose of 200 mg twice daily for at least 6 months were compared to a control group of 10 post-transplant subjects who were exposed only to environmental sources of fluoride (e.g., in drinking water and toothpaste). The levels of fluoride in the plasma of the group treated with 469 were found to be elevated relative to the control cohort (14.32 vs 2.54 μM; P < 0.001). Although renal clearance of fluoride is related to the glomerular filtration rate, renal function was not predictive of plasma fluoride levels. These concentrations remained significantly higher in the group treated with 469 even after adjusting for the levels of the immunosuppressive calcineurin inhibitors that are commonly used to treat transplant patients.(329) Half of the subjects treated with 469 showed evidence of periostitis. Two of these patients also had exostoses (periosteal bone growths), a known symptom of fluorosis caused by the strong affinity of fluoride for bones. In patients with evidence of periostitis, discontinuation of 469 relieved their pain and reduced the blood levels of both fluoride and alkaline phosphatase. Earlier studies of lung transplant patients exposed to 469 had also described cases of periostitis, although fluoride levels were not measured.(330,331) While studies of the metabolism of 469 had demonstrated that 5% of a dose of the drug was metabolized to release fluoride in humans (but not in mouse, rat, rabbit, guinea pig, or dog), those studies had failed to account for all of the excreted metabolites of the drug.(329,332,333) Collectively, these findings suggested that the adverse events described were elicited by the release of fluoride from 469 as a consequence of hepatic oxidative metabolism, leading to variably elevated fluoride levels in plasma after extended treatment. Metabolism studies identified the fluoropyrimidine moiety as one site of oxidative metabolism, while the difluorophenyl ring appeared to be inert.
7.2. Fluorinated Aromatics That Undergo Defluorination via Nucleophilic Substitution
In some chemical environments, aromatic C–F bonds may be subject to spontaneous or enzymatic ipso defluorination via an SNAr substitution reaction. An example of this phenomenon is provided by perfluorophenyl rings, which are predisposed by an excess of electron-withdrawing substituents to direct attack by nucleophiles such as GSH. Incubation of the DPP4 inhibitor 470 in GSH-enriched rat hepatocytes and liver microsomes led to the production of the four GSH conjugates 471–474.(334)
The formation of 471, a conjugate of undefined regiochemistry, occurred in phosphate buffer and was rationalized as the product of direct nucleophilic displacement of fluoride by GSH (Scheme 60, pathway a). Interestingly, the formation of 471 was enhanced in both phenobarbital- and dexamethasone-induced RLM, suggesting that GSTs, which are also induced by phenobarbital and dexamethasone in rats, may have been involved in catalysis.(334) The formation of 472–474 was NADPH-dependent and catalyzed by recombinant rat CYP3A1 and CYP3A2, although again, the regiochemistry of GSH adduction was speculative. The generation of 472 was hypothesized to occur either by oxidative defluorination of 471 or by a reaction involving the formation of the arene oxide 475, which captured GSH and eliminated fluoride to afford 476 as an initial intermediate (Scheme 60, pathway b). Reduction of the carbonyl group would give 477, and loss of HF would then afford 472. The quinones 478 and 479
were proposed as plausible metabolic precursors to 473 and 474, respectively, although alternative pathways to these metabolites were also considered.(334)
The formation of 471, a conjugate of undefined regiochemistry, occurred in phosphate buffer and was rationalized as the product of direct nucleophilic displacement of fluoride by GSH (Scheme 60, pathway a). Interestingly, the formation of 471 was enhanced in both phenobarbital- and dexamethasone-induced RLM, suggesting that GSTs, which are also induced by phenobarbital and dexamethasone in rats, may have been involved in catalysis.(334) The formation of 472–474 was NADPH-dependent and catalyzed by recombinant rat CYP3A1 and CYP3A2, although again, the regiochemistry of GSH adduction was speculative. The generation of 472 was hypothesized to occur either by oxidative defluorination of 471 or by a reaction involving the formation of the arene oxide 475, which captured GSH and eliminated fluoride to afford 476 as an initial intermediate (Scheme 60, pathway b). Reduction of the carbonyl group would give 477, and loss of HF would then afford 472. The quinones 478 and 479were proposed as plausible metabolic precursors to 473 and 474, respectively, although alternative pathways to these metabolites were also considered.(334)Scheme 60
Scheme 60. Proposed Metabolic Pathways of the Pentafluorophenyl-Based DPP4 Inhibitor 470 to give 471 and 472 (the Regiochemistry of GSH in 471 and 472 Was Not Determined)
Another pentafluorinated aromatic derivative that was reactive toward thiols was the β-tubulin inhibitor T138067 (480).(335−337)
This compound inactivated the β1, β2, and β4 isotype proteins by reacting with the conserved Cys239 to form a covalent adduct, resulting in microtubule degradation and cell-cycle arrest. The GSH conjugate 481 and derivatives 482 and 483 were isolated from tissue culture samples, from mouse, rat, dog, and human liver slices, and from the bile of mice treated intravenously with 480. The regiochemistry of fluorine displacement in 481 was established by 19F NMR analysis, which revealed only three resonance signal groups. Substitution was catalyzed in vitro by GSTα, GSTμ, and GSTπ, all of which are widely expressed in tissues, including those harvested from tumors. At physiological pH in the absence of GST enzymes, 480 did not react with GSH, indicating that the formation of 481 relied on metabolism and not just intrinsic reactivity.(335−337)
This compound inactivated the β1, β2, and β4 isotype proteins by reacting with the conserved Cys239 to form a covalent adduct, resulting in microtubule degradation and cell-cycle arrest. The GSH conjugate 481 and derivatives 482 and 483 were isolated from tissue culture samples, from mouse, rat, dog, and human liver slices, and from the bile of mice treated intravenously with 480. The regiochemistry of fluorine displacement in 481 was established by 19F NMR analysis, which revealed only three resonance signal groups. Substitution was catalyzed in vitro by GSTα, GSTμ, and GSTπ, all of which are widely expressed in tissues, including those harvested from tumors. At physiological pH in the absence of GST enzymes, 480 did not react with GSH, indicating that the formation of 481 relied on metabolism and not just intrinsic reactivity.(335−337)The 8-fluorotriazolopyridine-based c-Met inhibitors 484 and 485 were susceptible to displacement of fluorine by GSH in RLM, causing concern over the potential for GSH depletion and increased oxidative stress.(338) In addition, major metabolic pathways involving GSTs might lead to variable pharmacokinetics among human subjects due to differences in the expression of GST isoforms within the population. While removal of the C-8 fluorine atom in 484 and 485 would have avoided this metabolic pathway, this atom was an important contributor to potency. Hence, attention shifted toward modifying other parts of the molecule. It was hypothesized that the electrophilic character of the fluorinated triazolopyridine core could be attenuated by replacing the benzylic fluorine atoms with small alkyl groups and/or hydrogen atoms. This idea resulted in the identification of 486, which formed negligible amounts of GSH conjugates in bile-duct-cannulated rats and exhibited the best overall combination of potency and pharmacokinetic properties. The introduction of the 2-methoxyethoxy side chain also provided an alternative soft spot that redirected metabolism away from the core heterocycle in liver microsomes.(338)
7.3. 2-Fluoropyridine Derivatives
2-Fluoropyridine derivatives have been examined broadly in medicinal chemistry as a means of influencing pyridine basicity, interfering with biotransformation to the pyridine N-oxide, and reducing P450 inhibition and (when labeled with [18F]) for use in PET imaging.(339−345) Representative 2-fluoropyridine derivatives that have been advanced into clinical trials include the acetylcholine release enhancer DMP-543 (487), the AMPA receptor positive allosteric modulator 488, the CNS-penetrant phosphodiesterase-1 inhibitor 489, and the insulin-like growth factor receptor kinase inhibitor BMS-754807 (490).(346−348)
While 2-fluorinated pyridines have been exploited as reactive electrophilic intermediates in organic synthesis, there is little evidence to implicate intrinsic reactivity in biological systems as a problem. Nonetheless, their presence may result in the adoption of a cautionary stance.(349) Chemically, the hydrolytic loss of fluoride from 2-fluoropyridine derivatives can proceed via two mechanisms depending on the chemical environment: rings that are not activated by the presence of an electron-withdrawing substituent typically react via protonation of the ring N atom, whereas analogues substituted with strong electron-withdrawing substituents are more susceptible to SNAr substitution with expulsion of fluoride from the intermediate Meisenheimer complex.(350) While the reactivity of 2-fluoropyridine derivatives toward biological nucleophiles has not been studied in detail, the reactivity of a series of 2-chloropyridines toward ipso substitution by GSH in RLM, conditions that capture microsomal GST-1 activity, has been assessed and provides some guidance.(351) 2-Chloropyridine (491) was stable toward GSH in both the presence and absence of microsomal GST-1, with only low intrinsic reactivity observed (<5% transformed under the assay conditions). Indeed, appreciable SNAr reactivity catalyzed by microsomal GST-1 was observed only with the additional electronic activation afforded by nitro substitution of the ring (Table 3).(351) Thus, 2-chloro-3-nitropyridine (493) reacted with GSH in RLM at a rate of 236 nmol min–1 mg–1, translating to 100% depletion of the molecule over a 30 min incubation period. For comparison, 491 was consumed at a rate of less than 2 nmol min–1 mg–1 and 1-chloro-2-nitrobenzene (492) at a rate of 6.8 nmol min–1 mg–1. Other electron-withdrawing substituents at C-3 (494, 495) promoted the SNAr reaction, although less effectively than the nitro substituent. As might be anticipated, the electron-donating substituents in 496–499 mitigated the chemical reactivity. Notably, the background reactivity rate of 493 was low but measurable, with approximately 5% converted to the adduct in phosphate buffer, suggesting that modification of 2-halopyridines was driven primarily by metabolism.(351)
While 2-fluorinated pyridines have been exploited as reactive electrophilic intermediates in organic synthesis, there is little evidence to implicate intrinsic reactivity in biological systems as a problem. Nonetheless, their presence may result in the adoption of a cautionary stance.(349) Chemically, the hydrolytic loss of fluoride from 2-fluoropyridine derivatives can proceed via two mechanisms depending on the chemical environment: rings that are not activated by the presence of an electron-withdrawing substituent typically react via protonation of the ring N atom, whereas analogues substituted with strong electron-withdrawing substituents are more susceptible to SNAr substitution with expulsion of fluoride from the intermediate Meisenheimer complex.(350) While the reactivity of 2-fluoropyridine derivatives toward biological nucleophiles has not been studied in detail, the reactivity of a series of 2-chloropyridines toward ipso substitution by GSH in RLM, conditions that capture microsomal GST-1 activity, has been assessed and provides some guidance.(351) 2-Chloropyridine (491) was stable toward GSH in both the presence and absence of microsomal GST-1, with only low intrinsic reactivity observed (<5% transformed under the assay conditions). Indeed, appreciable SNAr reactivity catalyzed by microsomal GST-1 was observed only with the additional electronic activation afforded by nitro substitution of the ring (Table 3).(351) Thus, 2-chloro-3-nitropyridine (493) reacted with GSH in RLM at a rate of 236 nmol min–1 mg–1, translating to 100% depletion of the molecule over a 30 min incubation period. For comparison, 491 was consumed at a rate of less than 2 nmol min–1 mg–1 and 1-chloro-2-nitrobenzene (492) at a rate of 6.8 nmol min–1 mg–1. Other electron-withdrawing substituents at C-3 (494, 495) promoted the SNAr reaction, although less effectively than the nitro substituent. As might be anticipated, the electron-donating substituents in 496–499 mitigated the chemical reactivity. Notably, the background reactivity rate of 493 was low but measurable, with approximately 5% converted to the adduct in phosphate buffer, suggesting that modification of 2-halopyridines was driven primarily by metabolism.(351)Table 3. Reactivity of Halogenated Aromatic and Heteroaromatic Rings toward GSH in RLMa
| compd | X | R | reactivity rate (nmol min–1 mg–1)b | substrate depletionc | ΔG⧧ (kcal/mol) |
|---|---|---|---|---|---|
| 491 | N | H | <2 | 39.6 | |
| 492 | CH | NO2 | 6.8 ± 1.1 | 24.6 | |
| 493 | N | NO2 | 236 ± 8 | 100 ± 0 | 19.6 |
| 494 | N | CF3 | 31 ± 6 | 30.0 | |
| 495 | N | Cl | 26 ± 4 | 33.9 | |
| 496 | N | OCH3 | 17 ± 8 | 40.8 | |
| 497 | N | CH3 | 14 ± 5 | 40.9 | |
| 498 | N | NH2 | <5 | 39.7 | |
| 499 | N | OH | <5 | 32.6 |
a
Substrate (50 μM) was incubated with RLM (1 mg of protein/mL), GSH (5 mM), and MgCl2 (3 mM) in phosphate buffer (0.1 M; pH 7.4) at 37 °C. (Note: no NADPH was used in these experiments.) ΔG⧧ represents the calculated difference in Gibbs free energy between the ground state of the substrate and a Meisenheimer complex transition state derived by addition of thiolate.
b
Incubations were of varied duration (1 to 40 min).
c
Samples were incubated for 30 min; the data presented are the percentage decreases of the substrate, and the values are means ± SD (n = 3).
Linopirdine (500), the predecessor to 487, was subject to N-oxidation in rats, dogs, and humans to afford the N-oxides 501 and 502 (Scheme 61).(352−354) The fluorinated derivative 503 was explored for its potential to resist this metabolic pathway. Synthetic studies with 503 failed to produce N-oxides despite the use of strong oxidizing reagents that included CF3CO3H, suggesting considerable intrinsic resistance toward oxidation and, by extension, downstream conjugative metabolism. N-Oxidation enhanced the reactivity of 2-halopyridines, and 2-fluoropyridine 1-oxide reacted with dipeptides at room temperature via displacement of fluoride by the terminal amino moiety.(355,356) Thus, the occurrence of the N-oxidation pathway in vivo for 2-halopyridines would create concern over the potential for generation of reactive metabolites.
Scheme 61
Scheme 61. Formation of N-Oxide Metabolites 501 and 502 from 500 In Vivo
DMP-543 (487) emerged from an effort seeking to improve on the properties of the oxindole 500. The fluorine atoms were included to improve brain exposure and reduce the propensity for N-oxide formation that had been observed in studies of 500. While 487 was advanced into clinical trials as a potential treatment for Alzheimer’s disease, its high potency resulted in a low projected human dose of 25 μg, which presented unique challenges in the identification of a stable and uniform formulation.(357) Although the pure form of 487 was stable for several years, the tablet formulation was characterized by degradation to the monopyridone 504 (relative abundance of 0.5%) upon storage for 6 months at 40 °C and 75% relative humidity. The chemical degradation was dependent on the temperature, pH, and excipient composition but not the drug concentration. The likely degradation mechanism was informed by prior solution stability studies that had revealed sequential hydrolysis of the fluorine substituents under acidic conditions but good stability for 51 days at 80 °C in phosphate (pH 6.6) and borate (pH 9.2) buffers. These observations prompted the suggestion that the addition of a basic excipient might enhance the chemical stability.(358) This approach was effective, as a revised formulation that included 1% magnesium stearate (a base and lubricant) and lactose monohydrate (a diluent) suffered <0.1% degradation under the same storage conditions described above.
The discovery of 488 was informed by experience with the lead compound 505, which was characterized by acceptable potency and receptor activation but poor brain penetration. 
The latter issue was attributed to the high polar surface area (PSA) of 92 Å2 and efflux by P-gp, problems that could not be resolved by manipulation of the methanesulfonamide moiety.(346) The pyridine analogue 506 exhibited considerably weaker binding affinity, but its activation of AMPA receptors was comparable to that of cyclothiazide, a reference standard. While 506 demonstrated the targeted low PSA (59 Å2) and clogP (2.3) values, it also exhibited moderate clearance in RLM and potent inhibition of CYP1A2 (IC50 = 600 nM). The targeted physicochemical parameters limited the scope of substitution of the pyridine ring to halogen and alkyl substituents, and the 2- and 6-fluoro compounds 507 and 508 emerged as compounds with preferred profiles. This substitution pattern addressed the CYP1A2 inhibition and slightly increased the clogP value from 2.3 to 2.6 while preserving a low PSA.(346) Resolution of 508 revealed that biological activity resided with the S isomer 488 (pEC50 = 5.6, asym max = 107%), which demonstrated low turnover in RLM and HLM. The high membrane permeability of 488 coupled with an absence of efflux by P-gp contributed to the observed 61% oral bioavailability in rats and good CNS penetration, properties that subtended its efficacy in rat models of cognition. Metabolism studies of 488 in rat, dog, monkey, and human liver microsomes revealed five minor metabolites, of which three resulted from hydroxylation of the indane ring and two from hydroxylation of the isopropyl and aryl moieties (Figure 12). No metabolic changes to the fluoropyridine ring were described, and there was no apparent evidence of genotoxicity in vitro. In clinical studies, the pharmacokinetic profile of 488 revealed rapid absorption and a surprisingly long apparent half-life of 107 to 168 h.(346)
The latter issue was attributed to the high polar surface area (PSA) of 92 Å2 and efflux by P-gp, problems that could not be resolved by manipulation of the methanesulfonamide moiety.(346) The pyridine analogue 506 exhibited considerably weaker binding affinity, but its activation of AMPA receptors was comparable to that of cyclothiazide, a reference standard. While 506 demonstrated the targeted low PSA (59 Å2) and clogP (2.3) values, it also exhibited moderate clearance in RLM and potent inhibition of CYP1A2 (IC50 = 600 nM). The targeted physicochemical parameters limited the scope of substitution of the pyridine ring to halogen and alkyl substituents, and the 2- and 6-fluoro compounds 507 and 508 emerged as compounds with preferred profiles. This substitution pattern addressed the CYP1A2 inhibition and slightly increased the clogP value from 2.3 to 2.6 while preserving a low PSA.(346) Resolution of 508 revealed that biological activity resided with the S isomer 488 (pEC50 = 5.6, asym max = 107%), which demonstrated low turnover in RLM and HLM. The high membrane permeability of 488 coupled with an absence of efflux by P-gp contributed to the observed 61% oral bioavailability in rats and good CNS penetration, properties that subtended its efficacy in rat models of cognition. Metabolism studies of 488 in rat, dog, monkey, and human liver microsomes revealed five minor metabolites, of which three resulted from hydroxylation of the indane ring and two from hydroxylation of the isopropyl and aryl moieties (Figure 12). No metabolic changes to the fluoropyridine ring were described, and there was no apparent evidence of genotoxicity in vitro. In clinical studies, the pharmacokinetic profile of 488 revealed rapid absorption and a surprisingly long apparent half-life of 107 to 168 h.(346)Figure 12
Figure 12. Metabolic soft spots associated with 488 in liver microsomes.
The potent PDE-1 inhibitor ITI-214 (489) (Ki PDE-1 = 5.8 pM) is being evaluated clinically for its potential to address movement disorders, including Parkinson’s disease, and the cognitive deficits associated with Alzheimer’s disease, schizophrenia, and attention deficit hyperactivity disorder.(347) Compound 489 exhibited higher log D values (4.41 at pH 7.4 and 1.27 at pH 1.7) than the non-fluorinated variant 509 (3.54 at pH 7.4 and −0.58 at pH 1.7), 
reflecting the reduced basicity of the pyridine nitrogen. While this difference was accompanied by reduced solubility, the oral pharmacokinetics of 489 in mice and rats was superior to that of 509, with a 10-fold higher plasma area under the curve (AUC) in mice and a 20-fold advantage in CNS penetration. Although preclinical profiling studies of 489 have been described, details of the metabolism have yet to be disclosed.(359)
reflecting the reduced basicity of the pyridine nitrogen. While this difference was accompanied by reduced solubility, the oral pharmacokinetics of 489 in mice and rats was superior to that of 509, with a 10-fold higher plasma area under the curve (AUC) in mice and a 20-fold advantage in CNS penetration. Although preclinical profiling studies of 489 have been described, details of the metabolism have yet to be disclosed.(359)A medicinal chemistry effort exploring 4-aryl-N-arylcarbonyl-2-aminothiazoles as Hec1/Nek2 inhibitors, which exhibited antiproliferative activity against several cancer cell lines, offered the opportunity to compare the properties of the unsubstituted pyridine derivative 510 with those of the 2-fluoro analogue 511.(360)
While the in vitro potencies of the two compounds toward the MB231 breast cancer cell line were similar, 511 was characterized by 2-fold lower clearance and an almost 2-fold higher AUC following intravenous administration to rats. The low clearance of 511 in vivo reflected low metabolic clearance in RLM, in which the half-life was approximately 340 min. However, following oral administration to rats (20 mg/kg) in a vehicle of 1% methylcellulose in H2O, the bioavailability of 511 (4.6%) was lower than that of 510 (12.4%). This difference was attributed to the lower basicity and aqueous solubility of 511 (190 nM in phosphate buffer containing 1% DMSO at pH 7) relative to the unsubstituted pyridine analogues. This problem was solved by formulating 511 in a mixture of 5% DMSO, 10% cremophor, and 85% H2O, which increased the oral bioavailability in the rat to 22.7%, a result consistent with solubility-limited absorption.(360)
While the in vitro potencies of the two compounds toward the MB231 breast cancer cell line were similar, 511 was characterized by 2-fold lower clearance and an almost 2-fold higher AUC following intravenous administration to rats. The low clearance of 511 in vivo reflected low metabolic clearance in RLM, in which the half-life was approximately 340 min. However, following oral administration to rats (20 mg/kg) in a vehicle of 1% methylcellulose in H2O, the bioavailability of 511 (4.6%) was lower than that of 510 (12.4%). This difference was attributed to the lower basicity and aqueous solubility of 511 (190 nM in phosphate buffer containing 1% DMSO at pH 7) relative to the unsubstituted pyridine analogues. This problem was solved by formulating 511 in a mixture of 5% DMSO, 10% cremophor, and 85% H2O, which increased the oral bioavailability in the rat to 22.7%, a result consistent with solubility-limited absorption.(360)Analogous to the circumstance with the 2-fluoropyridine rings, there is scant information available on the potential for problems with 4-fluoropyridine derivatives. However, some 4-chloro- and 4-bromopyridines have been studied, which may allow inference about potential chemical reactivity and metabolism. These compounds are usually stable during incubations with GSH under ambient conditions, but SNAr substitution of the halide can be catalyzed by GST. The 4-halopyridines 512 and 513
were discovered by fragment screening to be MBIs of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that regulates nitric oxide production by catabolizing Nω,Nω-dimethyl-l-arginine, an endogenous inhibitor of nitric oxide synthases.(361) Mutation data supported a mechanism of inhibition that was dependent on the reaction of these compounds with the catalytic Cys249 of DDAH via an SNAr process, in which the pyridinium form of the molecule was stabilized in the active site by the proximal Asp66 (Scheme 62). More precise details of the mechanism were discerned by a combination of X-ray crystallographic data and modeling studies, which further illuminated the role of Asp66. This analysis suggested the involvement of a catalytic triad (Cys249, Ser248, Glu65) that activated the thiol moiety of Cys249 of DDAH. The Cys249 residue was characterized by an unusually high pKa value of ∼8.8, reflecting the dependence on a catalytic triad.(362)
were discovered by fragment screening to be MBIs of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that regulates nitric oxide production by catabolizing Nω,Nω-dimethyl-l-arginine, an endogenous inhibitor of nitric oxide synthases.(361) Mutation data supported a mechanism of inhibition that was dependent on the reaction of these compounds with the catalytic Cys249 of DDAH via an SNAr process, in which the pyridinium form of the molecule was stabilized in the active site by the proximal Asp66 (Scheme 62). More precise details of the mechanism were discerned by a combination of X-ray crystallographic data and modeling studies, which further illuminated the role of Asp66. This analysis suggested the involvement of a catalytic triad (Cys249, Ser248, Glu65) that activated the thiol moiety of Cys249 of DDAH. The Cys249 residue was characterized by an unusually high pKa value of ∼8.8, reflecting the dependence on a catalytic triad.(362)Scheme 62
Scheme 62. Mechanism Proposed to Explain the Reaction of 513 with DDAH
The semisynthetic spectinamide antibiotics, represented generically by 514, are derived from spectinomycin (515) and exhibit activity toward multidrug-resistant tuberculosis strains. 
The 4-chloropyridine moiety of certain representatives of this class, including 516, were subject to GSH conjugation via an SNAr process in human and rat S9 fractions (Scheme 63).(363) The rates of GSH conjugation were dependent on both the species and the substitution pattern on the pyridine ring. The calculated pKa values of the pyridyl nitrogen atoms also correlated well with the extent of GSH displacement. Less basic pyridines were more stable, consistent with a mechanism involving enzymatic protonation of the heterocycle that set the stage for attack of GSH at C-4. Non-halogenated and C-3-halogenated pyridines were stable toward conjugation. Similarly, a 5-fluoro-4-chloro-substituted pyridine was also stable, a finding that was attributed to the reduced basicity of the heterocycle.(363)
The 4-chloropyridine moiety of certain representatives of this class, including 516, were subject to GSH conjugation via an SNAr process in human and rat S9 fractions (Scheme 63).(363) The rates of GSH conjugation were dependent on both the species and the substitution pattern on the pyridine ring. The calculated pKa values of the pyridyl nitrogen atoms also correlated well with the extent of GSH displacement. Less basic pyridines were more stable, consistent with a mechanism involving enzymatic protonation of the heterocycle that set the stage for attack of GSH at C-4. Non-halogenated and C-3-halogenated pyridines were stable toward conjugation. Similarly, a 5-fluoro-4-chloro-substituted pyridine was also stable, a finding that was attributed to the reduced basicity of the heterocycle.(363)Scheme 63
Scheme 63. Metabolism of 4-Chloropyridine-Based Spectinamide Derivatives 516 Catalyzed by GST
7.4. Fluorinated Purine Derivatives
GSH conjugates of 6-halopurines are substrates of the multidrug resistance-associated protein 1 transporter (MRP1) and serve as useful probes for the study of this efflux pump.(364−366) (MRP1 is encoded by the gene ABCC1 and should not be confused with P-gp, encoded by ABCB1.) The success of this approach, which is based on the metabolite extrusion method, depends on the rapid conversion of CNS-permeable 6-halopurines to their GSH conjugates in the CNS followed by transport of these metabolites back across the membrane. The labeled 6-bromo-7-methyl-7H-purine (523) (Table 4) showed early promise as a reagent for these studies. However, in order to identify an improved probe and explore differences among nonclinical species, the rates of reactivity of other 6-halo-7-methyl-7H-purines and 6-halo-9-methyl-9H-purines toward GSH were measured in phosphate buffer and in brain homogenates (mouse, rat, and rhesus monkey) that contain GSTs.(366−369) The results revealed that in both series, the fluoro derivatives (517 and 521) were more intrinsically reactive toward GSH in buffer than their higher halogen homologues 518–520 and 522–524, respectively, while the 6-halo-7-methyl-7H-purines 521–524 were more reactive than the 9-methyl isomers 517–520 (Table 4). Rates of conversion were higher in brain homogenate but varied by species. A high ratio of the rate of enzymatic to intrinsic displacement was considered to be advantageous in these studies, since it would minimize nonspecific displacement by thiols in vivo.(366,367)
Table 4. Intrinsic and GST-Catalyzed Rates of Reactivity between Halo-Substituted Purines and GSHa
a
knon is the rate constant for the non-enzymatic process in phosphate buffer (0.1 M, pH 7.4) supplemented with GSH (2 mM).
b
kenz is the rate constant for the enzyme-catalyzed process in cerebral cortical homogenate supplemented with GSH (2 mM).
The series of 6-halogenated 2′,3′-dideoxypurine (ddP) derivatives 525–528 was studied in an effort to identify a prodrug of 2′,3′-dideoxyinosine (didanosine, ddI, 529), a premise that would require these compounds to be recognized as substrates of adenosine deaminase (Table 5).(370) An experiment assessing the activities of these compounds toward HIV-1 infection in cell culture revealed that the trend of antiretroviral potencies in the series (F > Cl > Br > I) reflected the half-lives in whole blood (F < Cl < Br < I) (Table 5).(370,371) Additionally, the increased lipophilicity of these halogenated purines relative to 529 provided a means of selectively targeting the virus in the CNS, in which rates of conversion were 10-fold higher in brain homogenates than in whole blood, potentially offering improved treatment of HIV-1-related neurological abnormalities. The half-lives and rates of conversion in these matrices are provided in Table 5.(371) The 6-fluoro derivative 525 caused lethargy and labored breathing in rats and was not evaluated further, as the therapeutic margins were deemed to be too narrow. The 6-chloro analogue 526 provided a balance of properties, delivering substantially higher concentrations of 529 to the brain than an equivalent intravenous infusion of the parent nucleoside.
Table 5. Kinetics of Hydrolysis of 6-Halo ddP Analogues 525–528 by Adenosine Deaminase to Give 529 at 37 °C and Half-Lives in Blood and Brain Homogenate
| t1/2 (min) | |||||
|---|---|---|---|---|---|
| compd | X | Km (M) | Vmax (M/min) | blood | brain tissue |
| 525 | F | 7.2 × 10–4 | 1.4 × 10–4 | 3.5 | 0.46 |
| 526 | Cl | 0.017 | 4.4 × 10–5 | 270 | 19 |
| 527 | Br | 0.016 | 4.0 × 10–5 | 280 | 20 |
| 528 | I | ND | ND | 1690 | 178 |
| whole blooda | brain tissuea | ||||||
|---|---|---|---|---|---|---|---|
| compd | X | k1 (min–1) | k2 (min–1) | F | k1 (min–1) | k2 (min–1) | F |
| 525 | F | 0.15 | 0.13 | 0.54 | 1.51 | ∼0 | 1.00 |
| 526 | Cl | 3.0 × 10–3 | 7.4 × 10–3 | 0.28 | 0.032 | ∼0 | 1.00 |
a
k1 is the rate of conversion to ddI by adenosine deaminase. k2 is the rate of conversion to ddI by other unknown sources. F is the total fraction of prodrug converted to ddI.
A similar prodrug approach was explored in the context of 531–533, 6-substituted derivatives of acyclovir (530), and 535–537, 6-substituted derivatives ganciclovir (534), 
which were anticipated to be susceptible to conversion to the corresponding guanines by adenosine deaminase.(372−374) In both series, the fluoro derivatives (531 and 535) were superior substrates of the enzyme compared with either the chloro (532 and 536) or amino (533 and 537) analogues (Table 6). The isopropyl ester 538, a double prodrug of 530, provided at least 51% conversion to the parent drug following oral dosing to rats based on the recovery of 530 from urine over 48 h. This concept was extended to 540, which performed similarly as a prodrug of penciclovir (539) in vivo.(375)
which were anticipated to be susceptible to conversion to the corresponding guanines by adenosine deaminase.(372−374) In both series, the fluoro derivatives (531 and 535) were superior substrates of the enzyme compared with either the chloro (532 and 536) or amino (533 and 537) analogues (Table 6). The isopropyl ester 538, a double prodrug of 530, provided at least 51% conversion to the parent drug following oral dosing to rats based on the recovery of 530 from urine over 48 h. This concept was extended to 540, which performed similarly as a prodrug of penciclovir (539) in vivo.(375)Table 6. Kinetics of Hydrolysis of 6-Substituted Analogues by Adenosine Deaminase to Afford Either Acyclovir (530) or Ganciclovir (534)
In a structurally related purine-like chemotype, the mTor inhibitor 541 was relatively stable in HLM but metabolized in human hepatocytes to the GSH conjugate 542.(376) Studies of similar compounds also demonstrated biotransformation in hepatocytes at the same aromatic carbon atom, with the higher chloro- and bromo-substituted homologues also undergoing direct displacement by GSH and the lower hydrogen-substituted homologue experiencing oxidation by aldehyde oxidase. While the involvement of GST in the former displacement reaction was not investigated, the metabolite profiles of these compounds suggested a degree of reactivity that was sufficient to justify deprioritization of this series from further inquiry.(376)
7.5. Fluorinated Alkyl Substituents
Carmegliptin (543) is a potent and long-acting DPP4 inhibitor that was studied clinically for its potential to treat type-2 diabetes.(377,378) Early analogues in the series were characterized by a tricyclic aminobenzo[a]quinolizine scaffold and a structurally distinct S1 recognition motif, with the fluoromethyl-substituted analogue 544 shown to bind to human DPP4 by X-ray crystallography.(377) This compound also offered 10-fold improved inhibition (IC50 = 0.5 nM) over the corresponding CH3 analogue, suggesting that potency and properties in the aryl series could be modulated through optimization of the S1 substituent. Although 544 exhibited the best fit in the S1 pocket, this compound was found to be Ames-positive and clastogenic in the micronucleus test.(377,378) Installing a more polar pyridine ring in the S1 ligand position led to 545, a potent DPP4 inhibitor with improved composite properties. However, additional benefits were realized by tuning the polarity across this S1 substituent, resulting in 543. Uncharacteristic of drugs in this class, 543 was found to undergo both renal and hepatic excretion, suggesting that it might be useful in patients with renal impairment. The metabolism of 543 does not appear to involve any specific changes to the fluoromethyl group, and while phase 1 and 2 clinical studies indicated its suitability as a safe oral antidiabetic agent with once-daily administration, development was terminated.(379)
The [18F]-monofluoromethyl-substituted thiazole SP203 (546) was developed as a radioligand for imaging of mGluR5 receptors in the brain.(380) However, in both the rat and monkey, radioactivity was observed in bone, indicating release of [18F]fluoride. To explore the mechanism of defluorination, both [18F]-546 and its non-radiolabeled analogue were administered to rats, and brain homogenates and urine were analyzed for metabolites. In those studies, radio-HPLC was used to detect [18F]fluoride as a major metabolite in both matrices. In a subsequent incubation with brain homogenate ex vivo, 546 exhibited a half-life of only 20 min and was converted to a major metabolite, whereas no such metabolism was apparent in whole blood ex vivo. Analysis of the brain extract by LC–MS/MS identified the GSH conjugate 547 and the N-acetylcysteine derivative 548. Only the latter was detected in rat urine, indicative of complete processing of 547 along the mercapturic acid pathway. Thus, GSH conjugation of the 2-fluoromethyl group of 546 was responsible for defluorination in rats.(380)
The bioactivation of aryl fluoromethyl and difluoromethyl groups can be leveraged for the design of MBIs of enzymes, an approach that has been explored in the context of inhibitors of tyrosine hydroxylase.(381) Tyrosine hydroxylation is the rate-limiting step in catecholamine biosynthesis, and α-methyl-l-tyrosine (metyrosine, 549) 
is a tyrosine hydroxylase inhibitor that is approved for the treatment of hypertensive patients who have a pheochromocytoma, a rare adrenal tumor associated with elevated catecholamine levels.(382)dl-2-Fluoromethyl-p-tyrosine (550) and dl-2-difluoromethyl-p-tyrosine (551) were designed as MBIs of tyrosine hydroxylase on the premise that oxidation of these compounds to a phenol by the enzyme would set the stage for loss of HF and the generation of reactive quinone methides (Scheme 64).(381) Both 550 and 551 were characterized as competitive inhibitors of purified bovine adrenal tyrosine hydroxylase. However, additional biochemical data have not been reported, and it remains unclear whether these compounds do indeed act by the intended pathway.
is a tyrosine hydroxylase inhibitor that is approved for the treatment of hypertensive patients who have a pheochromocytoma, a rare adrenal tumor associated with elevated catecholamine levels.(382)dl-2-Fluoromethyl-p-tyrosine (550) and dl-2-difluoromethyl-p-tyrosine (551) were designed as MBIs of tyrosine hydroxylase on the premise that oxidation of these compounds to a phenol by the enzyme would set the stage for loss of HF and the generation of reactive quinone methides (Scheme 64).(381) Both 550 and 551 were characterized as competitive inhibitors of purified bovine adrenal tyrosine hydroxylase. However, additional biochemical data have not been reported, and it remains unclear whether these compounds do indeed act by the intended pathway.Scheme 64
Scheme 64. Postulated Mechanism of Inactivation of Tyrosine Hydroxylase by 550 and 551
While benzylic fluorides are chemically more reactive than the difluoromethyl and trifluoromethyl homologues toward substitution reactions, there are circumstances where aryl difluoromethyl and trifluoromethyl moieties are reactive.(27) For example, the non-enzymatic hydrolysis of 6-difluoromethyltryptophan (552) to 6-formyltryptophan (553) 
occurred slowly (k = 0.0039 min–1) under neutral conditions.(383) However, the reaction was accelerated 104-fold when the pH was raised to deprotonate the indole NH (Scheme 65, path a). In this series, the solution stability of homologous 6-substituted indoles increased in the order of CH2F < CHF2 ≪ CF3. Tryptophanase, a PLP-dependent enzyme that converts 552 to 555, apparently did not facilitate this reaction since there was evidence of protein-dependent conversion of the difluoromethyl group. Rather, tryptophanase activated the aromatic ring by protonation at C-3, leading to the release of the 6-(difluoromethyl)-1H-indole (555) (Scheme 65, path b), a process that would preclude anion formation and fluoride elimination. Hence, the enzyme catalyzed the catabolism of 552 in much the same manner as it processes tryptophan, leading to the formation of 555 and 556 via the PLP-derivative 554. The latter complex was then subject to hydrolytic decomposition, yielding pyruvic acid (420) and NH3.(383) However, this pathway required the heterocyclic system of the substrate to be electron-rich, thereby slowing turnover for substrates carrying an electron-withdrawing group at C-6.
occurred slowly (k = 0.0039 min–1) under neutral conditions.(383) However, the reaction was accelerated 104-fold when the pH was raised to deprotonate the indole NH (Scheme 65, path a). In this series, the solution stability of homologous 6-substituted indoles increased in the order of CH2F < CHF2 ≪ CF3. Tryptophanase, a PLP-dependent enzyme that converts 552 to 555, apparently did not facilitate this reaction since there was evidence of protein-dependent conversion of the difluoromethyl group. Rather, tryptophanase activated the aromatic ring by protonation at C-3, leading to the release of the 6-(difluoromethyl)-1H-indole (555) (Scheme 65, path b), a process that would preclude anion formation and fluoride elimination. Hence, the enzyme catalyzed the catabolism of 552 in much the same manner as it processes tryptophan, leading to the formation of 555 and 556 via the PLP-derivative 554. The latter complex was then subject to hydrolytic decomposition, yielding pyruvic acid (420) and NH3.(383) However, this pathway required the heterocyclic system of the substrate to be electron-rich, thereby slowing turnover for substrates carrying an electron-withdrawing group at C-6.Scheme 65
Scheme 65. Base-Catalyzed Reaction (Path a) and Trytophanase-Mediated Metabolism (Path b) of 552
The reactivity of the 2-difluoromethylphenol moiety was leveraged in the design of MBIs of neuraminidases intended for the treatment of influenza virus infection.(384) These glycoside hydrolases cleave sialic acid moieties from glycoproteins and regulate viral replication. 2-Difluoromethylphenolic glycosides were first reported in the 1990s as glucosidase inhibitors, and similar approaches have been applied in the design of inhibitors of a range of glycosidases, including neuraminidases, galactosidases, N-acetyl glucosaminidase, and other hydrolases that have extended to phosphatases, sulfatases, and proteases. 2-Fluoromethylphenolic glycosides, represented by 557 (Scheme 66), were stable until the glycosidic bond was cleaved, and 4-substituted isomers behaved similarly. Following neuraminidase-mediated cleavage of 557, the resulting phenolate 558 was configured to eliminate fluoride, a reaction that yielded the o-quinone methide 559, which inactivated the enzyme by forming the covalent adduct 560.(384) Characterization of a library of synthetic 2-difluoromethylphenyl sialosides enabled the identification of potent and selective reagents that labeled neuraminidases according to this mechanism. Constituent members of these libraries differentially inhibited two neuraminidases, Vibrio cholerae neuraminidase and human neuraminidase 2, and the most potent inhibitors of each neuraminidase were selected for additional study. A kinetic analysis of these inhibitors demonstrated that synthetic modifications to the aglycone moiety resulted in improvements to the Ki with little change in other kinetic constants, including kinact and the half-life.
Scheme 66
Scheme 66. Mechanism-Based Inactivation of Neuraminidases by Sialoside Substrates Bearing Difluoromethylphenol Groups
Mechanism-based fluorescent labeling of β-galactosidase by 2-difluoromethylphenyl aglycones has also been demonstrated in the context of proteomics applications.(385) One such compound, 4-[5-(dimethylamino)naphthalene-1-sulfonamido]-2-(difluoromethyl)phenyl β-galactopyranoside (561) (Figure 13), was used to tag the catalytic sites of several bacterial galactosidases with a fluorescent dansylated aglycone that was captured using an antidansyl antibody and characterized by MALDI-TOF/TOF tandem mass spectrometry (Scheme 67). Galactosidase-mediated cleavage of the probe would release galactose (562) and the phenol 563 en route to the quinone methide 564, an intermediate with sufficient reactivity to capture a range of nucleophilic residues as adducts (565). Among the enzymes studied were the Xanthomonas manihotis fungal β-galactosidase, which reacted via the arginine residue found within the peptide fragment 56IPRAYWKD;(63)E. coli β-galactosidase, which was decorated at the catalytic glutamic acid residue Glu537; and Bacillus circulans β-galactosidase, which became labeled at Glu259. These results suggested that reactions with this inhibitor were influenced by both its spatial arrangement with respect to the catalytic cavity and the nucleophilicity of the amino acid residue. This methodology could be extended to the characterization of other retaining and inverting glycoside hydrolases.(385)
Figure 13
Figure 13. Design of the fluorescent probe 561 for assessment of galactosidase activity.
Scheme 67
Scheme 67. Mechanism-Based Labeling of a Galactosidase by the Fluorescent Reagent 561
Difluoromethyl substituents of imidazole rings are susceptible to base-catalyzed hydrolytic decomposition, and the rate of fluoride loss rises with increasing pH, consistent with the mechanism depicted in Scheme 68.(386) The half-lives for the conversion of 4-difluoromethylimidazole (566) and 2-difluoromethylimidazole (567) to the corresponding aldehydes 570 and 571 at physiological pH were slightly less than 3 h, largely precluding their application in drug discovery (Scheme 68). In contrast, the calculated half-life of 2-trifluoromethylimidazole (568) at pH 7.4 (corrected for a pKa of 10.11) was approximately 124 days, a result that illustrated the stabilizing effect of an additional fluorine atom on this substituted ring. Difluorohistidinol (569) struck a compromise in stability, with a half-life of approximately 11 h at pH 7.4.(386)
Scheme 68
Scheme 68. Mechanism of Base-Catalyzed Hydrolysis of Difluoromethylimidazoles 566 and 567 to Afford the Corresponding Aldehydes 570 and 571
Trifluoromethyl-substituted aromatic and heterocyclic rings are common motifs in drug design and are typically resistant to base-catalyzed hydrolysis.(1−8) However, an aryl trifluoromethyl moiety that is ortho or para to a hydroxyl or amino substituent can be labile under even mildly basic conditions. A mechanism to explain the hydrolysis of trifluoromethylphenols was elucidated in 1973 during stability studies of the C–F bonds of a number of compounds under weakly basic conditions.(387)o-Trifluoromethylphenol (572), p-trifluoromethylphenol (573), and 4-(3,3,3-trifluoroprop-1-en-1-yl)phenol (574) were hydrolyzed to the corresponding acids 575, 576, and 577 under these conditions. 
The kinetics of hydrolysis of 572 and 573 were consistent with a mechanism in which the o- and p-phenolates assisted in the displacement of fluoride to give difluoro-substituted quinone methide intermediates (Scheme 69). The stability of m-trifluoromethylphenol against hydroxide treatment substantiated the involvement of the oxyanions of 572 and 573 and argued against the kinetically equivalent direct displacement of fluoride from the neutral species of 572 and 573 by an SN2 process. When the olefin moiety of 574 was reduced, the CF3 group was stable in 1 N NaOH for 24 h, as would be anticipated for the mechanism presented in Scheme 69. These observations suggested the potential to design irreversible enzyme inhibitors in cases where the mode of catalysis would release a phenol derivative.
The kinetics of hydrolysis of 572 and 573 were consistent with a mechanism in which the o- and p-phenolates assisted in the displacement of fluoride to give difluoro-substituted quinone methide intermediates (Scheme 69). The stability of m-trifluoromethylphenol against hydroxide treatment substantiated the involvement of the oxyanions of 572 and 573 and argued against the kinetically equivalent direct displacement of fluoride from the neutral species of 572 and 573 by an SN2 process. When the olefin moiety of 574 was reduced, the CF3 group was stable in 1 N NaOH for 24 h, as would be anticipated for the mechanism presented in Scheme 69. These observations suggested the potential to design irreversible enzyme inhibitors in cases where the mode of catalysis would release a phenol derivative.Scheme 69
Scheme 69. Base-Catalyzed Hydrolysis of Phenol 572 to Give the Acid 575
Drug-related phototoxicity is a potential problem with compounds that absorb ultraviolet light, especially in the range of 290 to 400 nm, and occurs most frequently with compounds that are distributed into the skin or eyes.(388−390) The phototoxicity of a compound is determined experimentally in vivo; however, it may be estimated using key parameters including the maximal molar absorptivity and the energy difference between the lowest unoccupied and highest occupied molecular orbitals. In the case of CF3-substituted aromatic derivatives, absorption of ultraviolet light and electron elevation can lead to degradation and the generation of chemically reactive species.
Aryl CF3 substituents have also been shown to undergo photochemical degradation to acyl fluorides and carboxylic acids. The photochemical degradation of fluphenazine (578), a neuropsychiatric agent, was studied in the presence of peptide-based nucleophiles as part of an effort to understand the source of immune-related side effects that have been observed in therapy.(391,392) Both UVA and UVB light activated 578, with UVA penetration into peripheral capillaries providing an opportunity for photoactivation in vivo. This possibility was enhanced by the long plasma half-life of 578, which ranged from 15 h to several days. Both the triplet and singlet excited states of 578 were long-lived, and a photonucleophilic substitution of fluorine by H2O in the triplet state was proposed as the initial step (Scheme 70). This process afforded the electrophilic acyl fluoride 579, which was hydrolyzed by H2O to give the carboxylic acid (580, Nu = OH). Alternatively, reaction with biological nucleophiles gave rise to acylated adducts (580, Nu = protein).(392)
Scheme 70
Scheme 70. Mechanism of Photodegradation of the CF3 Moiety of 578
The selective serotonin reuptake inhibitor fluoxetine (581) was photoreactive in sunlit surface waters and was degraded in deionized H2O under simulated sunlight (half-life = 55.2 h).(393) The product of this reaction was identified as the carboxylic acid 582, confirming that H2O could act as the ultimate nucleophile in the absence of cellular material (Scheme 71).
Scheme 71
Scheme 71. Photodegradation of 581 under Simulated Sunlight in Deionized H2O to Give the Carboxylic Acid 582
Triflusal (583), a platelet-aggregation inhibitor prodrug used for the prevention and treatment of vascular thromboembolism, is associated with photoallergic side effects.(394) Metabolic deacylation of 583 afforded 2-hydroxy-4-trifluoromethylbenzoic acid (584), the pharmacologically active form, which was subject to hydrolytic photodegradation of the CF3 moiety to give 586 (Scheme 72).(394) The involvement of the triplet state in the photodegradation pathway was shown by laser flash photolysis and quenching experiments, and the binding of a photogenerated derivative of 584 to bovine serum albumin was demonstrated using UV/vis and fluorescence spectroscopy. Amino acids appeared to be the ultimate nucleophiles in this example, reacting with the acyl fluoride 585 to form covalent adducts 587 that were believed to initiate the photoallergic reaction.(394) The role of lysine residues in these reactions was revealed in a study exploring interactions between 585 and ubiquitin, a protein that contains seven Lys residues.(395) In that study, a combination of fluorescence spectroscopy, laser flash photolysis, and proteomic analysis demonstrated the formation of monoadducts 588 as the major products of this reaction. Interestingly, adduction occurred at all of the lysine residues of the protein.
Scheme 72
Scheme 72. Photodegradation of 584, Which Is Produced In Vivo from the Prodrug 583
Similarly, irradiation (310 nm) of 3,5-diamino-1-trifluoromethylbenzene (589) resulted in hydrolytic defluorination to give the carboxylic acid 590, presumably via reaction of an intermediate acyl fluoride with H2O (Scheme 73).(396) However, the presence of the aniline moieties in 589 and its products allowed for competing reactions of the acyl fluoride with the amines to produce dark-colored products including 591, 592, and 593. The progress of this reaction could be monitored by measuring the pH of the solution, which fell from 6.2 to 3.2 as the reaction proceeded.
Scheme 73
Scheme 73. Light-Catalyzed Polymerization of 589
Photoactivation of the CF3 moieties of 4-trifluoromethylphenols to generate latent electrophiles has been explored as a means of targeting lysine residues of proteins.(397) 4-Trifluoromethyl-1-naphthol (594) and 4′-(trifluoromethyl)-[1,1′-biphenyl]-4-ol (598) were activated to quinone methide intermediates 595 and 599, respectively, upon irradiation and formed adducts 597 and 601 when photolyzed in the presence of human serum albumin. These reactions were proposed to proceed by way of the imidoyl fluorides 596 and 600 as intermediates (Scheme 74).(397) Additional characterization of these complexes demonstrated chemoselectivity of the electrophilic species toward specific lysine residues within albumin, as 594 reacted covalently with Lys106 and Lys414 located in subdomains IA and IIIA, respectively, and 599 bound to Lys195 in subdomain IIA. Docking and molecular dynamics simulations provided insight into the molecular basis of the selectivity for these subdomains. These studies suggested a broader opportunity to silence target proteins by designing ligands that could be activated with light to covalently modify constituent lysine residues.(397)
Scheme 74
Scheme 74. Use of Aryl CF3 Moieties in the Design of Latent Electrophiles, Which Can Be Generated by Irradiation with Light and Used to Covalently Modify Target Proteins
2-Methyl-1,4-naphthoquinone (menadione, 602) is a subversive substrate of both human and Plasmodium GSH reductases, and the latter is critical to the survival of Plasmodium parasites amid the high concentrations of reactive oxygen species that are present in erythrocytes.(398) Accordingly, these proteins are a validated antimalarial target. 
Atovaquone (605), which is used in conjunction with the antifolate proguanil, is an inhibitor of the Plasmodium parasite electron transport chain that acts on the mitochondrial cytochrome bc1 complex 3 to interfere with redox cycling and affect the redox equilibrium in infected red blood cells. Because 605 was based on the same naphthoquinone core structure as 602 and lawsone (603), the concept of structural hybridization was explored in an attempt to identify a single compound that could disrupt both functions simultaneously.(398) Trifluoromenadione (604) acted as a prodrug of 603 in which the CF3 moiety was eliminated via a vinylogous haloform-type reaction with two potential underlying mechanisms. In Scheme 75, path a, the Michael addition of H2O to the CF3-substituted carbon atom of 604 set the stage for elimination of CF3– to give 603, the purported mechanism of prodrug activation. The alternative mechanism presented in Scheme 75, path b, envisaged Michael addition of H2O to the more electrophilic carbon atom of the quinone moiety of 604, affording an intermediate enolate that would require tautomerization of the α-hydroxy ketone intermediate to expel CF3– and give 603. The design principle anticipated that 606 would act as a prodrug of 605 in vivo following path a, since path b would be feasible only for R ≠ H.(398) However, 605 did not function as a prodrug of 604, indicating that path b was the likely mode of conversion of 604 to 603.
Atovaquone (605), which is used in conjunction with the antifolate proguanil, is an inhibitor of the Plasmodium parasite electron transport chain that acts on the mitochondrial cytochrome bc1 complex 3 to interfere with redox cycling and affect the redox equilibrium in infected red blood cells. Because 605 was based on the same naphthoquinone core structure as 602 and lawsone (603), the concept of structural hybridization was explored in an attempt to identify a single compound that could disrupt both functions simultaneously.(398) Trifluoromenadione (604) acted as a prodrug of 603 in which the CF3 moiety was eliminated via a vinylogous haloform-type reaction with two potential underlying mechanisms. In Scheme 75, path a, the Michael addition of H2O to the CF3-substituted carbon atom of 604 set the stage for elimination of CF3– to give 603, the purported mechanism of prodrug activation. The alternative mechanism presented in Scheme 75, path b, envisaged Michael addition of H2O to the more electrophilic carbon atom of the quinone moiety of 604, affording an intermediate enolate that would require tautomerization of the α-hydroxy ketone intermediate to expel CF3– and give 603. The design principle anticipated that 606 would act as a prodrug of 605 in vivo following path a, since path b would be feasible only for R ≠ H.(398) However, 605 did not function as a prodrug of 604, indicating that path b was the likely mode of conversion of 604 to 603.Scheme 75
Scheme 75. Two Potential Mechanisms for Elimination of –CF3 from 604
The 2-methyl-1,4-naphthoquinone derivative 607 and its 2-difluoromethyl analogue 608 were evaluated for inhibition of the water-borne blood fluke Schistosoma mansoni.(399)
The methyl-substituted quinone 607 inhibited S. mansoni thioredoxin-GSH reductase (SmTGR), a unique enzyme that protects the pathogen against the reactive oxygen species produced by the host immune response. The inactivation of SmTGR by 607 was time-dependent, forcing the enzyme into a futile cycle that culminated in the buildup of the reduced 3-phenoxymethyl variant 609 and the drawdown of reducing NADPH equivalents that would normally be used to detoxify reactive oxygen species (Scheme 76A). The inhibitory effect of 607 toward SmTGR translated to strong ex vivo antischistosomal activity, demonstrating a proof of concept for the target.(399) However, the inhibitory phenotype of the difluoromethyl analogue 608 toward SmTGR was qualitatively different in that the compound elicited polymerization and precipitation of the enzyme in vitro but failed to kill worms in culture. The different profile was explained by the tautomerization of 608 to the electrophilic o-quinone methide 611, which reacted with the enzyme in the biochemical assay to afford 612 rather than engaging in the redox cycling that would afford 610 (Scheme 76B). The lack of inhibitory activity of 608 ex vivo suggested that only redox-active molecules possessed the desired biochemical pharmacology. However, neither 607 nor 608 was active in vivo, an outcome attributed to poor bioavailability.(399)
The methyl-substituted quinone 607 inhibited S. mansoni thioredoxin-GSH reductase (SmTGR), a unique enzyme that protects the pathogen against the reactive oxygen species produced by the host immune response. The inactivation of SmTGR by 607 was time-dependent, forcing the enzyme into a futile cycle that culminated in the buildup of the reduced 3-phenoxymethyl variant 609 and the drawdown of reducing NADPH equivalents that would normally be used to detoxify reactive oxygen species (Scheme 76A). The inhibitory effect of 607 toward SmTGR translated to strong ex vivo antischistosomal activity, demonstrating a proof of concept for the target.(399) However, the inhibitory phenotype of the difluoromethyl analogue 608 toward SmTGR was qualitatively different in that the compound elicited polymerization and precipitation of the enzyme in vitro but failed to kill worms in culture. The different profile was explained by the tautomerization of 608 to the electrophilic o-quinone methide 611, which reacted with the enzyme in the biochemical assay to afford 612 rather than engaging in the redox cycling that would afford 610 (Scheme 76B). The lack of inhibitory activity of 608 ex vivo suggested that only redox-active molecules possessed the desired biochemical pharmacology. However, neither 607 nor 608 was active in vivo, an outcome attributed to poor bioavailability.(399)Scheme 76
Scheme 76. Substituent at the 2-Position of the Quinone (Methyl or Difluoromethyl) Affects the Chemical and Biochemical Pharmacological Profile of Antischistosomal Compounds
The CF3 moiety of 5-(trifluoromethyl)-2′-deoxyuridine (trifluridine, 613) is a critical determinant of the mechanism-based inhibition of thymidylate synthase that underlies its anticancer action.(400−402)
Thymidylate synthase catalyzes the methylation of 2-deoxyuridine monophosphate to give thymidine monophosphate, which as the only intracellular source of this essential DNA building block is critical to support the rapid division and proliferation of immortalized cells. Blocking of thymidylate synthase also leads to the accumulation of uridine monophosphate, increasing the potential for this base to be misincorporated into DNA. The enzymatic methylation catalyzed by thymidylate synthase proceeds via a ternary complex between the substrate uridine monophosphate, thymidylate synthase, and the cofactor 5,10-methylene tetrahydrofolic acid, which is the source of the methyl group installed in thymidine monophosphate. Inhibition of thymidylate synthase by 613 required metabolism to obtain the monophosphate 614, which bound to the active site in the absence of the cofactor and was accepted by the enzyme the same fashion as the natural substrate (Scheme 77). Thus, nucleophilic addition of the catalytic Cys198 to the β-position of the conjugated amide led to an anionic intermediate that was poised to eliminate fluoride from the CF3 moiety, affording the Michael acceptor 615.(402) Addition of the phenol of Tyr146 to the α,β-unsaturated amide 615 followed by elimination of a second fluoride from the adduct generated 616. Although 616 was more stable than 615, it was prone to hydrolytic degradation with ejection of the final fluoride, via 617 and 618, to produce the tyrosine ester 619. The elimination of Cys198 from 619 by a retro-Michael process produced the ester 620. The involvement of Cys198 and Tyr146 in this mechanism was confirmed by treatment of thymidylate synthase with 2′-[3H]-614 followed by proteolytic digestion and sequencing of the labeled peptides. Separately, X-ray crystallographic analysis provided evidence for a covalent bond between the nucleotide and Tyr146 in the enzyme–inhibitor complex. For oncology indications, 613 is typically administered in combination with an inhibitor of thymidine phosphorylase, which prevents the breakdown of 614 to trifluoromethyl thymine.(402)
Thymidylate synthase catalyzes the methylation of 2-deoxyuridine monophosphate to give thymidine monophosphate, which as the only intracellular source of this essential DNA building block is critical to support the rapid division and proliferation of immortalized cells. Blocking of thymidylate synthase also leads to the accumulation of uridine monophosphate, increasing the potential for this base to be misincorporated into DNA. The enzymatic methylation catalyzed by thymidylate synthase proceeds via a ternary complex between the substrate uridine monophosphate, thymidylate synthase, and the cofactor 5,10-methylene tetrahydrofolic acid, which is the source of the methyl group installed in thymidine monophosphate. Inhibition of thymidylate synthase by 613 required metabolism to obtain the monophosphate 614, which bound to the active site in the absence of the cofactor and was accepted by the enzyme the same fashion as the natural substrate (Scheme 77). Thus, nucleophilic addition of the catalytic Cys198 to the β-position of the conjugated amide led to an anionic intermediate that was poised to eliminate fluoride from the CF3 moiety, affording the Michael acceptor 615.(402) Addition of the phenol of Tyr146 to the α,β-unsaturated amide 615 followed by elimination of a second fluoride from the adduct generated 616. Although 616 was more stable than 615, it was prone to hydrolytic degradation with ejection of the final fluoride, via 617 and 618, to produce the tyrosine ester 619. The elimination of Cys198 from 619 by a retro-Michael process produced the ester 620. The involvement of Cys198 and Tyr146 in this mechanism was confirmed by treatment of thymidylate synthase with 2′-[3H]-614 followed by proteolytic digestion and sequencing of the labeled peptides. Separately, X-ray crystallographic analysis provided evidence for a covalent bond between the nucleotide and Tyr146 in the enzyme–inhibitor complex. For oncology indications, 613 is typically administered in combination with an inhibitor of thymidine phosphorylase, which prevents the breakdown of 614 to trifluoromethyl thymine.(402)Scheme 77
Scheme 77. Mechanism of Inhibition of Thymidylate Synthase by 614, the Monophosphate Metabolite of 613
While CF3 substituents can be useful in modulating compound properties, polyfluorination tends to increase the lipophilicity since the π coefficient for fluorine is 0.14.(1−5) Accordingly, each additional CF3 adds 0.42 units to the log P value. However, matched molecular pair analysis has indicated that in the absence of specific effects, up to five fluorine atoms can be installed in a molecule without a significant effect on P-gp-mediated efflux, permeability, lipophilicity, and metabolic stability.(403) Nevertheless, it is important to deploy the CF3 moiety and other lipophilic groups deliberately and with care in order to maintain the lipophilicity within a drug-like range. The cholesteryl ester transfer protein (CETP) inhibitor anacetrapib (621) 
offered a notable example of how fluorination could be used to obtain a desirable pharmacological profile but concurrently how its overuse could have unintended consequences. There were three CF3 groups present in 621, and these moieties were metabolically inert despite the presence of other soft spots.(404) To date, 621 has been the only CETP inhibitor to successfully complete phase 3 clinical trials, in which it decreased the plasma concentration of low-density lipoprotein, increased the concentration of high density lipoprotein, and reduced cardiovascular events in patients with atherosclerotic disease.(405) However, regulatory filings for 621 were not pursued despite a profile that suggested acceptable efficacy and safety. This decision was due in part to the extraordinarily long terminal pharmacokinetic half-life of 621.(406) In patients who had taken 621 for 18 months, measurable systemic exposure to the drug continued for up to 4 years after the suspension of therapy. Follow-up studies seeking to illuminate this phenomenon revealed extensive distribution of 621 into adipose tissue and lipid droplets in a fashion not dependent on lipase activity or active transport.(407) In retrospect, this behavior is perhaps unsurprising since 621 was characterized by a log D value of 7.1, a high level of lipophilicity that may have helped facilitate interactions with the target protein in the lipoprotein particle. It is instructive to recognize that this pharmacokinetic behavior was not presaged by an especially large steady-state volume of distribution in preclinical species after a single dose (1.1 ± 0.6 L/kg in rats and 0.3 ± 0.1 L/kg in monkeys), perhaps because much of the drug was retained in circulation as the result of an association with plasma proteins (the free fraction was <0.5% in both species). Similarly, a majority of the radioactivity (approximately 80% in rats and 90% in monkeys) was recovered within 48 h after administration of a single oral dose of [14C]-621.(404) This pharmacokinetic profile was consistent with a slow but extensive distribution into adipose tissue, creating a depot from which the drug could escape slowly over a long period of time.(408) Hence, the lipophilicity afforded by CF3 groups should be deployed with care, and the pharmacokinetics of highly lipophilic compounds should be evaluated in multidose studies as a precaution to characterize the extent of accumulation into fatty tissue.
offered a notable example of how fluorination could be used to obtain a desirable pharmacological profile but concurrently how its overuse could have unintended consequences. There were three CF3 groups present in 621, and these moieties were metabolically inert despite the presence of other soft spots.(404) To date, 621 has been the only CETP inhibitor to successfully complete phase 3 clinical trials, in which it decreased the plasma concentration of low-density lipoprotein, increased the concentration of high density lipoprotein, and reduced cardiovascular events in patients with atherosclerotic disease.(405) However, regulatory filings for 621 were not pursued despite a profile that suggested acceptable efficacy and safety. This decision was due in part to the extraordinarily long terminal pharmacokinetic half-life of 621.(406) In patients who had taken 621 for 18 months, measurable systemic exposure to the drug continued for up to 4 years after the suspension of therapy. Follow-up studies seeking to illuminate this phenomenon revealed extensive distribution of 621 into adipose tissue and lipid droplets in a fashion not dependent on lipase activity or active transport.(407) In retrospect, this behavior is perhaps unsurprising since 621 was characterized by a log D value of 7.1, a high level of lipophilicity that may have helped facilitate interactions with the target protein in the lipoprotein particle. It is instructive to recognize that this pharmacokinetic behavior was not presaged by an especially large steady-state volume of distribution in preclinical species after a single dose (1.1 ± 0.6 L/kg in rats and 0.3 ± 0.1 L/kg in monkeys), perhaps because much of the drug was retained in circulation as the result of an association with plasma proteins (the free fraction was <0.5% in both species). Similarly, a majority of the radioactivity (approximately 80% in rats and 90% in monkeys) was recovered within 48 h after administration of a single oral dose of [14C]-621.(404) This pharmacokinetic profile was consistent with a slow but extensive distribution into adipose tissue, creating a depot from which the drug could escape slowly over a long period of time.(408) Hence, the lipophilicity afforded by CF3 groups should be deployed with care, and the pharmacokinetics of highly lipophilic compounds should be evaluated in multidose studies as a precaution to characterize the extent of accumulation into fatty tissue.8. Fluorinated Sulfides, Sulfoxides, and Sulfones
ARTICLE SECTIONS
The SCF3 substituent offers unique properties but has not been highly represented in compounds evaluated clinically. The stimulatory amphetamine derivative (±)-tiflorex (flutiorex, 622), the anthelmintic agent monepantel (623), and the coccidiostat toltrazuril (624) are examples of fluorinated sulfides that have been studied clinically, while the dihydroorotate dehydrogenase inhibitor 625 has been examined in a preclinical setting. 
The SCF3 substituent is lipophilic (π = 1.44(254,320)), contributing to the distribution of SCF3-substituted compounds into fatty tissue. This pattern was apparent during a small clinical trial of 622 that explored its potential as an anorectic agent.(409,410) This study found that the concentration of 622 in erythrocytes was 4-to-8-fold higher than that in plasma over a time course of 6 h. Moreover, urinary elimination of 622 remained measurable at 48 h postadministration, suggesting low clearance and/or extensive distribution. This finding was despite the observation that urinary excretion of unchanged 622 over 24 h represented only a small fraction of the dose administered.(409,410)
The SCF3 substituent is lipophilic (π = 1.44(254,320)), contributing to the distribution of SCF3-substituted compounds into fatty tissue. This pattern was apparent during a small clinical trial of 622 that explored its potential as an anorectic agent.(409,410) This study found that the concentration of 622 in erythrocytes was 4-to-8-fold higher than that in plasma over a time course of 6 h. Moreover, urinary elimination of 622 remained measurable at 48 h postadministration, suggesting low clearance and/or extensive distribution. This finding was despite the observation that urinary excretion of unchanged 622 over 24 h represented only a small fraction of the dose administered.(409,410)The CF3S moiety is susceptible to oxidative metabolism to afford both the sulfoxide and the sulfone, metabolites that have been observed with 622–625. Oxidation to the sulfoxide may be characterized by a degree of enantioselectivity depending on the substrate and the enzyme involved, which is typically either a member of the P450 superfamily or an FMO. For example, biotransformation studies of 624 in RLM revealed that the relative rates of sulfoxidation to give the enantiomers 626 and 627 (the absolute configurations of these two sulfoxides are not known) could be manipulated by pretreating rats with different inducing agents prior to the harvesting of microsomes (Scheme 78).(411) Since the P450 enzymes induced by these agents were known, it was possible to identify certain inducible enzymes that catalyzed the formation of 626 (CYP1A1/CYP1A2) and others that favored the opposite enantiomer 627 (CYP2B1/CYP2B2). Consistent with this finding, pretreatment of rats with 3-methylcholanthrene, an inducer of CYP1A, prior to administration of 624 led to dramatic changes in the plasma AUCs of the sulfoxide enantiomers. Specifically, the AUC of 626 increased by approximately 3-fold, whereas the AUC of 627 declined to only 14% of the value in the vehicle control.(412) The second step in this metabolic sequence, oxidation of the sulfoxide enantiomers 626 and 627 to the achiral sulfone 628, was also stereoselective (Scheme 78). Although both of these reactions were catalyzed by CYP3A, the rate of oxidation was 7-fold higher for 626. Hence, the exposure of each discrete sulfoxide enantiomer was a composite function of the rate and enantioselectivity of the reactions governing its formation and clearance, both of which were oxidative processes.
Scheme 78
Scheme 78. Oxidative Metabolism of the CF3S Substituent of 624 in RLM and Rats to Afford the Sulfoxides 626 and 627 and Sulfone 628a
aThe absolute configurations of the sulfoxides 626 and 627 were not determined, and the assignments shown are arbitrary.
Oxidative metabolism typically facilitates the disposition of drugs, rendering products that are more hydrophilic and more susceptible to transfer into bile and urine. However, the metabolic sequence described above tends to result in the formation of sulfone metabolites that exhibit longer half-lives in vivo than the parent sulfides. For example, the plasma half-life of 622 following intravenous administration to anesthetized rats was 7.4 h, but the half-lives of the sulfone derived from 622 and its N-dealkyated metabolite were 2.6 and 9.3 h, respectively.(413) Remarkably, the plasma AUC of the sulfone derived from 622 in this experiment was approximately 12-fold higher than the AUC of the sulfide 622. Analogous trends were noticed in a series of β-hydroxy enamides, in which a comparison of a CF3S-substituted compound to its CF3SO2 metabolite revealed the latter to have a 7-fold longer half-life in the rat.(234) The anthelmintic drug 623 was also converted to a biologically active sulfone metabolite exhibiting a larger steady-state volume of distribution, lower clearance, and increased mean residence time compared with 623.(414) Similarly, the half-life of 628 was longer than that of 624 in pigs (Scheme 78).(415) The common theme in these examples is that although oxidation of the CF3S moiety to the sulfone increases its hydrophilicity (the π coefficient for the CF3SO2 moiety is 0.55 compared with 1.44 for CF3S, and both are more lipophilic than CH3SO2, for which π = −1.63), these metabolites retain significant lipophilicity that renders them prone to tissue penetration.(320) These properties may imbue the sulfone with reduced clearance and/or increased distribution relative to the sulfide, both of which would work to lengthen the half-life in vivo. Thus, it should be anticipated that a CF3S substituent will be susceptible to metabolism to the sulfoxide and sulfone derivatives in vivo and that those metabolites may represent the main drug-related compounds circulating in the blood. However, it should be emphasized that CF3S substituents may not necessarily confer reduced metabolic stability relative to CF3O. This trend was exemplified by 630 and 632, the homologues of pretomanid (629) and riluzole (631), respectively, in which the CF3O moieties were replaced by CF3S.(416) As captured in Table 7, 630 and 632 were characterized by rates of metabolic clearance in HLM that were similar to those of their respective CF3O-substituted homologues 629 and 631. These SCF3-substituted compounds also exhibited higher lipophilicity (log P) and lower aqueous solubility (pH 6.8) than their matched OCF3-substitued pairs, whereas no consistent trend was apparent with respect to plasma protein binding or membrane permeability.
Table 7. In Vitro Profiling Data for 629 and 631 and Their Respective CF3S Analogues 630 and 632
| compound | log P | aqueous solubility at pH 6.8 (mM) | MDCK LE Papp A-Ba | Clint (μL min–1 mg–1)b | human plasma protein binding |
|---|---|---|---|---|---|
| 629 | 2.2 | 0.008 | 21.1 | <25 | 38% |
| 630 | 2.7 | 0.005 | 15.5 | <25 | 60% |
| 631 | 3.2 | 0.986 | 118.3 | 38.9 | 85% |
| 632 | 3.6 | 0.124 | 152.0 | 40.9 | 76% |
a
Passive permeability in Madin–Darby canine kidney low-efflux cells, expressed as a ratio of concentrations.
b
Intrinsic clearance in HLM.
A similar sequence of metabolic reactions can be expected in connection with the use of α,α-difluoroethyl sulfides (CH3CF2S), which are also converted sequentially to sulfoxides and sulfones in a stereoselective manner.(253) These compounds are characterized by lower lipophilicity than the corresponding CF3S derivatives, a function of the dipole moment associated with the CF2 element, and therefore offer a potentially useful alternative in drug design.
The strongly electron-withdrawing aryl substituents CF3SO and CF3SO2 may confer unique properties to a molecule, projecting influence over metabolic pathways that introduce changes distal to the substituent. This concept was illustrated nicely by a study of substituent effects on the reactivity of 633, suicide inhibitors of phospholipase A2.(417) These inhibitors shared structural similarities with the native substrate of the enzyme and were hydrolyzed by phospholipase A2 to release the phospholipid portion of the molecule and the carboxylic acid 634 (Scheme 79). The chemical stability of 634 was directly related to the electron-withdrawing character of the R substituent, with strong electron-withdrawing groups like CF3SO2 stabilizing the negative charge on the phenolate of 635 and facilitating the formation of the reactive cyclic gem-dimethylglutaric anhydride 636. The rate of the cyclization step determined the extent of mechanism-based inhibition of phospholipase A2, with those substrates that cyclized slowly allowing diffusion of 634 away from the enzyme prior to release of the anhydride, resulting in nonspecific acylation.(417) However, rapid formation of the anhydride occurred mostly within the active site, increasing the chance of reaction with the enzyme. Consistent with this model, the empirical rate of enzyme inactivation (k) in this experiment was modeled with reasonable accuracy by the Hammett equation, log(k/k0) = σpρ, using σp constants measured previously for each of the phenyl substituents that were investigated. This inactivation rate increased as a function of the R group (CN < CH3SO2 < CF3SO < NO2 < CF3SO2), correlating approximately with the σp values, and the ρ value of 2.4 measured experimentally suggested a strong dependence on substituent effects. This example is a reminder that oxidative metabolism of an aryl SCF3 group may result in changes that strongly influence the electronics of the aryl ring and in extreme cases may lead to overall increases in chemical reactivity.
Scheme 79
Scheme 79. Substituent Effects on Phospholipase A2 Suicide Inhibition by 633
Although the COSCH2F motif is not used widely in medicinal chemistry, the glucocorticoid agonist fluticasone furoate (145) offers a visible example of the use of this moiety as a soft spot for oxidative metabolism.(13,161,418) Clinical administration of 145, a popular treatment for locally defined inflammatory disorders, is by inhalation for the treatment of asthma or by topical application for dermal indications. While 145 had a relatively long half-life of 15.3 h, driven in part by distribution into tissue (Vd = 642 L), extensive first-pass hepatic extraction limited the oral bioavailability to just 1.6%. Specifically, the rapid metabolism of 145 to the corresponding 17β-carboxylic acid 638, which was inactive as a glucocorticoid agonist, restricted the exposure of the compound in vivo and minimized the potential for systemic side effects (Scheme 80).(161) Studies using HLM demonstrated that the formation of 638 was inhibited by ketoconazole, an inhibitor of CYP3A, but not by selective chemical inhibitors of other P450 enzymes. Similarly, experiments using recombinant enzymes implicated CYP3A in metabolic turnover.(418) Collectively, these results framed the COSCH2F group as a limiting factor in the systemic exposure of 145 in humans. This molecule in essence acts as a soft drug, a salient point for those looking to revisit this feature in the design of medicines to be administered orally.(13)
Scheme 80
Scheme 80. Metabolism of the [(Fluoromethyl)thio]carbonyl Moiety of 145 by P450 to Afford the Carboxylic Acid 638
The pentafluorosulfanyl (SF5) substituent continues to attract the attention of the medicinal chemistry community, although there are few (if any) examples of applications that take full advantage of its unique properties.(419,420) In a study comparing the potential of several substituents to act as replacements for the tert-butyl moiety, the SF5 substituent conferred the highest aqueous solubility and lowest pKa among the homologues tested in addition to a log D value between those of tert-butyl and CF3. These results echoed a longstanding characterization of SF5 as being both highly polar and highly lipophilic.(421) Against this backdrop, several studies have examined substitution of CF3 by SF5 in a range of structural backgrounds in an attempt to improve on the composite properties of the CF3 derivatives.(422−425) The dihydroorotate dehydrogenase inhibitor DSM265 (639), an antimalarial agent, 
is the only compound bearing a SF5 substituent that has been advanced into clinical development.(426,427) Since malaria tends to afflict rural areas where access to health clinics may limit the delivery and quality of care, a medicine that can be administered easily and infrequently is highly advantageous. Compound 639 shows promise in this regard, exhibiting a long duration of action that may allow the drug to be administered as a single dose. It achieved an extended duration with a long pharmacokinetic half-life (86 to 118 h in humans) that was driven by low metabolic clearance, extensive distribution into tissue, and extensive binding to plasma proteins.(427) These characteristics clearly reflected the lipophilicity and metabolic stability of the SF5 substituent. Of the biotransformation pathways of 639 that have been characterized, none appeared to affect the SF5 moiety directly. It is also important to note that the preclinical studies of 639 revealed few inherent liabilities that might extend to other compounds with SF5 substituents.(426) Specifically, 639 did not induce P450, demonstrated little off-target activity in a broad pharmacology screen (ion channels, kinases, etc.), and exhibited marginal activity in patch-clamp studies that was offset by its low free fraction. Furthermore, preclinical safety studies showed that high doses of 639 were tolerated across preclinical species and that neither 639 nor the embedded aniline (4-pentafluoro-λ6-sulfanylaniline) gave rise to revertants in the Ames mutagenicity assay. With respect to issues to be resolved in the development of 639, one of the main challenges relates to its pharmaceutics properties and the apparent need for an enabled formulation. For the first-in-human study, 639 was formulated as a spray-dried dispersion that required suspension in a vehicle on-site, a process that was deemed to be cumbersome.(427) Hence, more work along these lines is indicated to identify a viable solid dosage form or other formulation suitable for use in remote areas.
is the only compound bearing a SF5 substituent that has been advanced into clinical development.(426,427) Since malaria tends to afflict rural areas where access to health clinics may limit the delivery and quality of care, a medicine that can be administered easily and infrequently is highly advantageous. Compound 639 shows promise in this regard, exhibiting a long duration of action that may allow the drug to be administered as a single dose. It achieved an extended duration with a long pharmacokinetic half-life (86 to 118 h in humans) that was driven by low metabolic clearance, extensive distribution into tissue, and extensive binding to plasma proteins.(427) These characteristics clearly reflected the lipophilicity and metabolic stability of the SF5 substituent. Of the biotransformation pathways of 639 that have been characterized, none appeared to affect the SF5 moiety directly. It is also important to note that the preclinical studies of 639 revealed few inherent liabilities that might extend to other compounds with SF5 substituents.(426) Specifically, 639 did not induce P450, demonstrated little off-target activity in a broad pharmacology screen (ion channels, kinases, etc.), and exhibited marginal activity in patch-clamp studies that was offset by its low free fraction. Furthermore, preclinical safety studies showed that high doses of 639 were tolerated across preclinical species and that neither 639 nor the embedded aniline (4-pentafluoro-λ6-sulfanylaniline) gave rise to revertants in the Ames mutagenicity assay. With respect to issues to be resolved in the development of 639, one of the main challenges relates to its pharmaceutics properties and the apparent need for an enabled formulation. For the first-in-human study, 639 was formulated as a spray-dried dispersion that required suspension in a vehicle on-site, a process that was deemed to be cumbersome.(427) Hence, more work along these lines is indicated to identify a viable solid dosage form or other formulation suitable for use in remote areas.Conclusion
ARTICLE SECTIONS
The ever-deepening understanding of the unique attributes of fluorine, coupled with the development of synthetic methodologies that are expanding access to new and useful fluorinated motifs, has led to the extensive deployment of this element in drug design. The judicious use of fluorine in a molecule can affect the physical, biochemical, pharmacological, and pharmacokinetic properties in ways that can be beneficial. The high strength and large dipole moment of the C–F bond, along with the strong electronegativity, small size, and modest lipophilicity of fluorine all subtend a versatility in drug design that is unmatched by any other element. However, while fluorine has often been used to enhance metabolic stability, studies of fluorinated compounds have uncovered metabolic pathways in which the fluorine atom influences the metabolic fate, informing the need to exercise care in its deployment. Despite its strength, there are many examples of C–F bond rupture during metabolism. This aspect of fluorine chemistry has been recognized for some time and specifically exploited in the design of enzyme inhibitors. However, other motifs are emerging that can be problematic in a more cryptic sense. The biotransformation of fluorinated compounds can set the stage for the departure of fluoride as a leaving group, sometimes generating chemically reactive intermediates. There are several other examples in which fluorinated molecules are subject to multistep metabolic pathways, resulting in the release of low-molecular-weight fluorinated toxins like 2-fluoroethanol, fluoroacetaldehyde, fluoroacetic acid, and fluorolactic acid. While fluorine is only modestly more lipophilic than a hydrogen atom, its use as part of a CF3 or SF5 moiety adds significantly to the lipophilicity of a compound. It has been suggested that up to five fluorine atoms can be installed as hydrogen atom replacements with minimal impact, but more heavily fluorinated compounds may present challenges with respect to drug distribution and disposition if global lipophilicity is not controlled. Nevertheless, the attributes of fluorine are such that its prominence in drug design will continue to grow. A collective view of the emerging data on the metabolism and disposition of fluorinated compounds suggests that some care should be exercised in its deployment in drug candidates.
Author Information
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- Benjamin M. Johnson - Pharmaceutical Candidate Optimization, Bristol Myers Squibb Company, 100 Binney Street, Cambridge, Massachusetts 02142, United States;
http://orcid.org/0000-0001-5277-4743;
- Nicholas A. Meanwell - Discovery Chemistry Platforms, Small Molecule Drug Discovery, Bristol Myers Squibb Company, Route 206 and Province Line Road, Princeton, New Jersey 08543, United States;http://orcid.org/0000-0002-8857-1515
Biographies
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Benjamin M. Johnson
Benjamin M. Johnson received his Ph.D. from the University of Illinois at Chicago, where he learned to use liquid chromatography–tandem mass spectrometry in the study of natural products. Since 2004 he has been at Bristol Myers Squibb (BMS), where he oversees an analytical drug-metabolism laboratory and leads project teams working to discover medicines for cancer and autoimmune disorders. He was involved in the discovery of daclatasvir (the first-in-class HCV NS5A inhibitor), rimegepant and vazegepant (calcitonin-gene-related peptide receptor antagonists), GSK-3532795/BMS-955176 (an HIV-1 maturation inhibitor), and LX9211 (an adaptor associated protein kinase 1 inhibitor) in addition to other compounds in clinical development. After hours, he is an avid runner and home cook.
Yue-Zhong Shu
Yue-Zhong Shu received his Ph.D. from the University of Toyama in Japan and conducted his Ph.D. and postdoctoral studies at Virginia Polytechnic Institute and State University in natural product chemistry and biotransformation. He joined BMS in 1992, where he became the leader of the natural product chemistry group. Over the past 20 years, he has led biotransformation teams supporting drug discovery in the fields of virology, neuroscience, metabolic diseases, oncology, immunology, cardiovascular disease, and fibrosis. His expertise in drug metabolism spans a range of synthetic molecules and nontraditional modalities, including antibody–drug conjugates, macrocyclic peptides, and protein degradation agents. He has been involved with many BMS drug candidates advanced to clinical development and to the marketplace.
Xiaoliang Zhuo
Xiaoliang Zhuo received his Ph.D. in toxicology from State University of New York at Albany and performed postdoctoral research at the University of Pennsylvania before joining BMS in 2004. He supports discovery research and clinical development across therapeutic areas, with a focus on the characterization and optimization of ADMET properties. These efforts have helped to advance several molecules with optimized profiles to clinical evaluation, including the HCV NS5B inhibitor beclabuvir. Since becoming a Diplomate of the American Board of Toxicology in 2014, he has investigated mechanisms of liver injury induced by clinical candidates and developed strategies to assess and mitigate the off-target risk. He enjoys writing research articles and has published papers on novel biotransformation pathways.
Nicholas A. Meanwell
Nicholas A. Meanwell received his Ph.D. from the University of Sheffield and conducted postdoctoral studies at Wayne State University before joining BMS in 1982. He has been associated with the discovery of BMY-433771, an inhibitor of respiratory syncytial virus fusion, the HIV-1 attachment inhibitor temsavir/fostemsavir, the HIV-1 maturation inhibitor GSK-3532795/BMS-955176, and the marketed HCV inhibitors asunaprevir (NS3), daclatasvir (NS5A), and beclabuvir (NS5B). He is the corecipient of a 2014 PhRMA Research and Hope Award for Biopharmaceutical Industry Research and a 2017 ACS Heroes of Chemistry Award. He was the recipient of the 2015 Philip S. Portoghese Medicinal Chemistry Lectureship Award and was inducted into the ACS Division of Medicinal Chemistry Hall of Fame in 2015.
| Abbreviations Used | |
| ADH | aldehyde dehydrogenase |
| ALDH | alcohol dehydrogenase |
| CB | cannabinoid receptor |
| CETP | cholesteryl ester transfer protein |
| CFC | chlorofluorocarbon |
| CRF | corticotropin-releasing factor |
| CNS | central nervous system |
| DAAO | d-amino acid oxidase |
| DDAH | dimethylarginine dimethylaminohydrolase |
| ddI | 2,3-dideoxyinosine |
| ddP | 2,3-dideoxypurine |
| DPP4 | dipeptidyl peptidase-4 |
| FMO | flavin-containing monooxygenase |
| GABA | γ-aminobutyric acid |
| GABAAT | γ-aminobutyric acid aminotransferase |
| GSH | glutathione |
| GST | glutathione S-transferase |
| GSTZ | glutathione S-transferase zeta |
| HCFC | hydrochlorofluorocarbon |
| HCV | hepatitis C virus |
| HFC | hydrofluorocarbon |
| HLM | human liver microsomes |
| ip | intraperitoneal |
| iv | intravenous |
| KSP | kinesin spindle protein |
| LC | liquid chromatography |
| MAO | monoamine oxidase |
| MBI | mechanism-based inhibitor |
| MDR | multidrug resistance protein 1 |
| MS | mass spectrometry |
| NAD | nicotinamide adenine dinucleotide |
| PDE | phosphodiesterase |
| PET | positron emission tomography |
| P-gp | P-glycoprotein |
| PLP | pyridoxal 5′-phosphate |
| RLM | rat liver microsomes |
| SAHase | S-adenosyl-l-homocysteine hydrolase |
| SM | squalene monooxygenase |
| SmTGR | thioredoxin-glutathione reductase |
| SSAO | semicarbazide-sensitive amine oxidase |
| SULT | sulfotransferase |
| TDI | time-dependent inhibition |
| UDPGA | uridine diphosphate glucuronic acid |
| UGT | UDP-glucuronosyltransferase |
| VAP-1 | vascular adhesion protein-1 |
References
ARTICLE SECTIONS
This article references 427 other publications.
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- 4Bohm, H. J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.; Muller, K.; Obst-Sander, U.; Stahl, M. Fluorine in medicinal chemistry. ChemBioChem 2004, 5, 637– 643, DOI: 10.1002/cbic.200301023[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2c3it1egug%253D%253D&md5=fa5ca2cfc278b83c80d5c96b18dfe312Fluorine in medicinal chemistryBohm Hans-Joachim; Banner David; Bendels Stefanie; Kansy Manfred; Kuhn Bernd; Muller Klaus; Obst-Sander Ulrike; Stahl MartinChembiochem : a European journal of chemical biology (2004), 5 (5), 637-43 ISSN:1439-4227.Fluorinated compounds are synthesized in pharmaceutical research on a routine basis and many marketed compounds contain fluorine. The present review summarizes some of the most frequently employed strategies for using fluorine substituents in medicinal chemistry. Quite often, fluorine is introduced to improve the metabolic stability by blocking metabolically labile sites. However, fluorine can also be used to modulate the physicochemical properties, such as lipophilicity or basicity. It may exert a substantial effect on the conformation of a molecule. Increasingly, fluorine is used to enhance the binding affinity to the target protein. Recent 3D-structure determinations of protein complexes with bound fluorinated ligands have led to an improved understanding of the nonbonding protein-ligand interactions that involve fluorine.
- 5Muller, K.; Faeh, C.; Diederich, F. Fluorine in pharmaceuticals: looking beyond intuition. Science 2007, 317, 1881– 1886, DOI: 10.1126/science.1131943[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2srnvVOnuw%253D%253D&md5=f624ab3733f7fc9d4b1711f2f3566ca2Fluorine in pharmaceuticals: looking beyond intuitionMuller Klaus; Faeh Christoph; Diederich FrancoisScience (New York, N.Y.) (2007), 317 (5846), 1881-6 ISSN:.Fluorine substituents have become a widespread and important drug component, their introduction facilitated by the development of safe and selective fluorinating agents. Organofluorine affects nearly all physical and adsorption, distribution, metabolism, and excretion properties of a lead compound. Its inductive effects are relatively well understood, enhancing bioavailability, for example, by reducing the basicity of neighboring amines. In contrast, exploration of the specific influence of carbon-fluorine single bonds on docking interactions, whether through direct contact with the protein or through stereoelectronic effects on molecular conformation of the drug, has only recently begun. Here, we review experimental progress in this vein and add complementary analysis based on comprehensive searches in the Cambridge Structural Database and the Protein Data Bank.
- 6Zhou, Y.; Wang, J.; Gu, Z.; Wang, S.; Zhu, W.; Acena, J. L.; Soloshonok, V. A.; Izawa, K.; Liu, H. Next generation of fluorine-containing pharmaceuticals, compounds currently in phase II-III clinical trials of major pharmaceutical companies: New structural trends and therapeutic areas. Chem. Rev. 2016, 116, 422– 518, DOI: 10.1021/acs.chemrev.5b00392[ACS Full Text], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVGms7o%253D&md5=e3d07d070fcf5b69a54f70bdba99acd4Next Generation of Fluorine-Containing Pharmaceuticals, Compounds Currently in Phase II-III Clinical Trials of Major Pharmaceutical Companies: New Structural Trends and Therapeutic AreasZhou, Yu; Wang, Jiang; Gu, Zhanni; Wang, Shuni; Zhu, Wei; Acena, Jose Luis; Soloshonok, Vadim A.; Izawa, Kunisuke; Liu, HongChemical Reviews (Washington, DC, United States) (2016), 116 (2), 422-518CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. We focus herein on a new collection of 51 fluorine-contg. mols. that either were approved after 2011 by the corresponding agencies or still are on clin. development. These compds. cover a large no. of therapeutic areas and, on this occasion, we decided to classify them according to the no. of fluorine atoms or fluorinated moieties. In each case, the structures and modes of action of these new drugs are revised, with a particular focus on the role of fluorine in the biol. and pharmaceutical properties, when appropriate. Finally, representative synthetic routes are highlighted.
- 7Wang, J.; Sanchez-Rosello, M.; Acena, J. L.; del Pozo, C.; Sorochinsky, A. E.; Fustero, S.; Soloshonok, V. A.; Liu, H. Fluorine in pharmaceutical industry: fluorine-containing drugs introduced to the market in the last decade (2001–2011). Chem. Rev. 2014, 114, 2432– 2506, DOI: 10.1021/cr4002879[ACS Full Text], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFegsb7I&md5=07faac419cd090503e82c6f00d05c338Fluorine in Pharmaceutical Industry: Fluorine-Containing Drugs Introduced to the Market in the Last Decade (2001-2011)Wang, Jiang; Sanchez-Rosello, Maria; Acena, Jose Luis; del Pozo, Carlos; Sorochinsky, Alexander E.; Fustero, Santos; Soloshonok, Vadim A.; Liu, HongChemical Reviews (Washington, DC, United States) (2014), 114 (4), 2432-2506CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A historic review. As expected from the fluorine position on the periodic table of elements, it possesses some extreme properties, in particular, ultimate electronegativity and oxidn. potential. Therefore,elemental fluorine can not be prepd. by chem. reaction, and its isolation in 1886 by Henri Moissan required scientific ingenuity and great personal courage. His historic effort earned him a Noble Prize (1906), and the developed electrolysis method is still in use for industrial prodn. of fluorine gas. However, further development of fluorine chem. was extremely sluggish, pursued by a handful of experts capable of handling the violent gas using specially designed lab. equipment. Industrial-scale prodn. of fluorochems. dates back to late 1930s. Currently, there are about 200 pharmaceuticals containingfluorine, including the 40 new compds. discussed in this review. One may agree that the contribution of the past decade indicates a significant 20% increase in the no. of fluorinated drugs on the market.
- 8Ilardi, E. A.; Vitaku, E.; Njardarson, J. T. Data-mining for sulfur and fluorine: an evaluation of pharmaceuticals to reveal opportunities for drug design and discovery. J. Med. Chem. 2014, 57, 2832– 2842, DOI: 10.1021/jm401375q[ACS Full Text], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFyksb%252FE&md5=b684f76341311979d0100ed68cffe714Data-Mining for Sulfur and Fluorine: An Evaluation of Pharmaceuticals To Reveal Opportunities for Drug Design and DiscoveryIlardi, Elizabeth A.; Vitaku, Edon; Njardarson, Jon T.Journal of Medicinal Chemistry (2014), 57 (7), 2832-2842CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. Among carbon, hydrogen, oxygen, and nitrogen, sulfur and fluorine are both leading constituents of the pharmaceuticals that comprise our medicinal history. In efforts to stimulate the minds of both the general public and expert scientist, statistics were collected from the trends assocd. with therapeutics spanning 12 disease categories (a total of 1969 drugs) from our new graphical montage compilation: disease focused pharmaceuticals posters. Each poster is a vibrant display of a collection of pharmaceuticals (including structural image, Food and Drug Administration (FDA) approval date, international nonproprietary name (INN), initial market name, and a color-coded subclass of function) organized chronol. and classified according to an assocn. with a particular clin. indication. Specifically, the evolution and structural diversity of sulfur and the popular integration of fluorine into drugs introduced over the past 50 years are evaluated. The presented qual. conclusions in this article aim to promote innovative insights into drug development.
- 9Jeschke, P. The unique role of fluorine in the design of active ingredients for modern crop protection. ChemBioChem 2004, 5, 570– 589, DOI: 10.1002/cbic.200300833[Crossref], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXjvFeqtLw%253D&md5=1f684935456162dfcbccf8069a6fd397The unique role of fluorine in the design of active ingredients for modern crop protectionJeschke, PeterChemBioChem (2004), 5 (5), 570-589CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. The task of inventing and developing active ingredients with useful biol. activities requires a search for novel chem. substructures. This process may trigger the discovery of whole classes of chems. of potential com. interest. Similar biol. effects can often be achieved by completely different compds. However, compds. within a given structural family may exhibit quite different biol. activities depending on their interactions with different intracellular proteins like enzymes or receptors. By varying the functional groups and structural elements of a lead compd., its interaction with the active site of the target protein, as well as its physicochem., pharmacokinetic, and dynamic properties can be improved. In this context, the introduction of fluorine into active ingredients has become an important concept in the quest for a modern crop protection product with optimal efficacy, environmental safety, user friendliness, and economic viability. Fluorinated org. compds. represent an important and growing family of com. agrochems. A no. of recently developed agrochem. candidates represent novel classes of chem. compds. with new modes of action; several of these compds. contain new fluorinated substituents. However, the complex structure-activity relationships assocd. with biol.-active mols. mean that the introduction of fluorine can lead to either an increase or a decrease in the efficacy of a compd., depending on its changed mode of action, physicochem. properties, target interaction, or metabolic susceptibility and transformation. Therefore, it is still difficult to predict the sites in a mol. at which fluorine substitution will result in optimal desired effects.
- 10Jeschke, P. The unique role of halogen substituents in the design of modern agrochemicals. Pest Manage. Sci. 2010, 66, 10– 27, DOI: 10.1002/ps.1829[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFGrsb3K&md5=6c28bc5a824d268f64c33e05e9b63df8The unique role of halogen substituents in the design of modern agrochemicalsJeschke, PeterPest Management Science (2010), 66 (1), 10-27CODEN: PMSCFC; ISSN:1526-498X. (John Wiley & Sons Ltd.)A review. The past 30 years have witnessed a period of significant expansion in the use of halogenated compds. in the field of agrochem. research and development. The introduction of halogens into active ingredients has become an important concept in the quest for a modern agrochem. with optimal efficacy, environmental safety, user friendliness and economic viability. Outstanding progress has been made, esp. in synthetic methods for particular halogen-substituted key intermediates that were previously prohibitively expensive. Interestingly, there has been a rise in the no. of com. products contg. mixed' halogens, e.g. one or more fluorine, chlorine, bromine or iodine atoms in addn. to one or more further halogen atoms. Extrapolation of the current trend indicates that a definite growth is to be expected in fluorine-substituted agrochems. throughout the twenty-first century. A no. of these recently developed agrochem. candidates contg. halogen substituents represent novel classes of chem. compds. with new modes of action. However, the complex structure-activity relationships assocd. with biol. active mols. mean that the introduction of halogens can lead to either an increase or a decrease in the efficacy of a compd., depending on its changed mode of action, physicochem. properties, target interaction or metabolic susceptibility and transformation. In spite of modern design concepts, it is still difficult to predict the sites in a mol. at which halogen substitution will result in optimal desired effects. This review describes comprehensively the successful utilization of halogens and their unique role in the design of modern agrochems., exemplified by various com. products from Bayer CropScience coming from different agrochem. areas. Copyright © 2009 Society of Chem. Industry.
- 11Fujiwara, T.; O’Hagan, D. Successful fluorine-containing herbicide agrochemicals. J. Fluorine Chem. 2014, 167, 16– 29, DOI: 10.1016/j.jfluchem.2014.06.014[Crossref], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtFCgu7%252FM&md5=7fe60bc6faa07bfd629833ce8911c488Successful fluorine-containing herbicide agrochemicalsFujiwara, Tomoya; O'Hagan, DavidJournal of Fluorine Chemistry (2014), 167 (), 16-29CODEN: JFLCAR; ISSN:0022-1139. (Elsevier B.V.)A review. Of the herbicides licensed worldwide, currently around 25% contain at least one fluorine atom and many contain multiple fluorines in the form of difluoro- and trifluoromethyl groups. Fluorine-contg. compds. have made a significant contribution to the development of products for the agrochems. industry and many organofluorine entities have found stable market positions. In this review we highlight the most important fluorinated herbicides in terms of their global use. The compds. are grouped by mode of action. A synthesis route is described for each compd. although the synthesis presented may not actually be the industrial process.
- 12Jeschke, P. Latest generation of halogen-containing pesticides. Pest Manage. Sci. 2017, 73, 1053– 1066, DOI: 10.1002/ps.4540[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjs1Oqsrw%253D&md5=ba3907b014cc806b92985351db88b1b0Latest generation of halogen-containing pesticidesJeschke, PeterPest Management Science (2017), 73 (6), 1053-1066CODEN: PMSCFC; ISSN:1526-498X. (John Wiley & Sons Ltd.)Agriculture is confronted with enormous challenges, from prodn. of enough high-quality food to water use, environmental impacts and issues combined with a continually growing world population. Modern agricultural chem. has to support farmers by providing innovative agrichems., used in applied agriculture. In this context, the introduction of halogen atoms into an active ingredient is still an important tool to modulate the properties of new crop protection compds. Since 2010, around 96% of the launched products (herbicides, fungicides, insecticides/acaricides and nematicides) contain halogen atoms. The launched nematicides contain the largest no. of halogen atoms, followed by insecticides/acaricides, herbicides and fungicides. In this context, fungicides and herbicides contain in most cases fluorine atoms, whereas nematicides and insecticides contain in most cases 'mixed' halogen atoms, for example chlorine and fluorine. This review gives an overview of the latest generation of halogen-contg. pesticides launched over the past 6 years and describes current halogen-contg. development candidates. © 2017 Society of Chem. Industry.
- 13Swallow, S. Fluorine in medicinal chemistry. Prog. Med. Chem. 2015, 54, 65– 133, DOI: 10.1016/bs.pmch.2014.11.001[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MngtFSlsw%253D%253D&md5=eae9cd9be216b857ef1cbdb54f8dfddfFluorine in medicinal chemistrySwallow StevenProgress in medicinal chemistry (2015), 54 (), 65-133 ISSN:0079-6468.Since its first use in the steroid field in the late 1950s, the use of fluorine in medicinal chemistry has become commonplace, with the small electronegative fluorine atom being a key part of the medicinal chemist's repertoire of substitutions used to modulate all aspects of molecular properties including potency, physical chemistry and pharmacokinetics. This review will highlight the special nature of fluorine, drawing from a survey of marketed fluorinated pharmaceuticals and the medicinal chemistry literature, to illustrate key concepts exploited by medicinal chemists in their attempts to optimize drug molecules. Some of the potential pitfalls in the use of fluorine will also be highlighted.
- 14Neilson, A. H.; Allard, A.-S. Degradation and transformation of organic fluorine compounds. In Organofluorines; Neilson, A. H., Ed.; The Handbook of Environmental Chemistry, Vol. 3N; Springer: Berlin, 2002.
- 15Liang, T.; Neumann, C. N.; Ritter, T. Introduction of fluorine and fluorine-containing functional groups. Angew. Chem., Int. Ed. 2013, 52, 8214– 8264, DOI: 10.1002/anie.201206566[Crossref], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFCrs7bI&md5=1dafdf60409a792e78d946731f6cc2b9Introduction of Fluorine and Fluorine-Containing Functional GroupsLiang, Theresa; Neumann, Constanze N.; Ritter, TobiasAngewandte Chemie, International Edition (2013), 52 (32), 8214-8264CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. This Review gives a brief summary of conventional fluorination reactions, including those reactions that introduce fluorinated functional groups, and focuses on modern developments in the field.
- 16Yang, X.; Wu, T.; Phipps, R. J.; Toste, F. D. Advances in catalytic enantioselective fluorination, mono-, di-, and trifluoromethylation, and trifluoromethylthiolation reactions. Chem. Rev. 2015, 115, 826– 870, DOI: 10.1021/cr500277b[ACS Full Text], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhslylt73E&md5=7c625074fd176dbd55756b514f83c1efAdvances in Catalytic Enantioselective Fluorination, Mono-, Di-, and Trifluoromethylation, and Trifluoromethylthiolation ReactionsYang, Xiaoyu; Wu, Tao; Phipps, Robert J.; Toste, F. DeanChemical Reviews (Washington, DC, United States) (2015), 115 (2), 826-870CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review on advances in catalytic enantioselective fluorination, mono-, di-, and trifluoromethylation, and trifluoromethylthiolation reactions.
- 17Ahrens, T.; Kohlmann, J.; Ahrens, M.; Braun, T. Functionalization of fluorinated molecules by transition-metal-mediated C–F bond activation to access fluorinated building blocks. Chem. Rev. 2015, 115, 931– 972, DOI: 10.1021/cr500257c[ACS Full Text], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvVWnsrvP&md5=10d7aaadbe8d77ed2bbe180823cde41cFunctionalization of Fluorinated Molecules by Transition-Metal-Mediated C-F Bond Activation To Access Fluorinated Building BlocksAhrens, Theresia; Kohlmann, Johannes; Ahrens, Mike; Braun, ThomasChemical Reviews (Washington, DC, United States) (2015), 115 (2), 931-972CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review that deals with transition-metal-mediated C1F bond activations which involve a cleavage of a C-F bond in polyfluorinated mols. and its transformation into carbon-element bonds, excluding hydrodefluorination or defluorination reactions.
- 18Nenajdenko, V. G.; Muzalevskiy, V. M.; Shastin, A. V. Polyfluorinated ethanes as versatile fluorinated C2-building blocks for organic synthesis. Chem. Rev. 2015, 115, 973– 1050, DOI: 10.1021/cr500465n[ACS Full Text], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVChsr4%253D&md5=9d9171191cc523457da69a5905b7205fPolyfluorinated Ethanes as Versatile Fluorinated C2-Building Blocks for Organic SynthesisNenajdenko, Valentine G.; Muzalevskiy, Vasiliy M.; Shastin, Aleksey V.Chemical Reviews (Washington, DC, United States) (2015), 115 (2), 973-1050CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Prepn. and reactions of polyfluorinated ethanes are addressed. Fluorinated derivs. of alkanes, alkenes, ethers, sulfides, carboxylic acids, sulfinic and sulfonic acids, alcs. and ketones, and arom. and heteroarom. compds. were efficiently prepd. using cheap and available starting materials.
- 19Yerien, D. E.; Barata-Vallejo, S.; Postigo, A. Difluoromethylation reactions of organic compounds. Chem. - Eur. J. 2017, 23, 14676– 14701, DOI: 10.1002/chem.201702311[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlOrt7rK&md5=848967a175fb629358d812640eab147bDifluoromethylation Reactions of Organic CompoundsYerien, Damian E.; Barata-Vallejo, Sebastian; Postigo, AlChemistry - A European Journal (2017), 23 (59), 14676-14701CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. The difluoromethylation strategies to functionalize different families of org. compds., for the late-stage introduction of a CF2H or CF2Y moieties into org. substrates was presented. These methodologies include, (i) a metal-photoredox catalysis; (ii) transition metal-catalyzed thermal protocols; and (iii) transition-metal-free strategies.
- 20Wang, X.; Wang, X.; Wang, J. Application of carbene chemistry in the synthesis of organofluorine compounds. Tetrahedron 2019, 75, 949– 964, DOI: 10.1016/j.tet.2019.01.016[Crossref], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVWjtb0%253D&md5=c218876889b4e665512cf4b3f67f8867Application of carbene chemistry in the synthesis of organofluorine compoundsWang, Xuyong; Wang, Xi; Wang, JianboTetrahedron (2019), 75 (8), 949-964CODEN: TETRAB; ISSN:0040-4020. (Elsevier Ltd.)A review. Carbenes are in general highly reactive species, though which various transformations are possible. Carbene chem. has been recently explored in the development of new methodologies for the synthesis of organofluorine compds. This short report summarizes the recent advances in this area.
- 21Park, B. K.; Kitteringham, N. R. Effects of fluorine substitution on drug metabolism: pharmacological and toxicological implications. Drug Metab. Rev. 1994, 26, 605– 643, DOI: 10.3109/03602539408998319[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXmslSqtbc%253D&md5=a766e575c62e197c3a03a4557273b9ddEffects of fluorine substitution on drug metabolism: Pharmacological and toxicological implicationsPark, B. Kevin; Kitteringham, Neil R.Drug Metabolism Reviews (1994), 26 (3), 605-43CODEN: DMTRAR; ISSN:0360-2532.A review with 112 refs. Physicochem. properties of fluorinated drugs, fluorine substitution effect on drug metab., and fluorinated compds. as enzyme inhibitors are discussed.
- 22Park, B. K.; Kitteringham, N. R.; O’Neill, P. M. Metabolism of fluorine-containing drugs. Annu. Rev. Pharmacol. Toxicol. 2001, 41, 443– 470, DOI: 10.1146/annurev.pharmtox.41.1.443[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjsVaqtrc%253D&md5=0e2ee242da0f4cef8d203e92aee03180Metabolism of fluorine-containing drugsPark, B. Kevin; Kitteringham, Neil R.; O'Neill, Paul M.Annual Review of Pharmacology and Toxicology (2001), 41 (), 443-470CODEN: ARPTDI; ISSN:0362-1642. (Annual Reviews Inc.)A review with 109 refs. This article reviews current knowledge of the metab. of drugs that contain fluorine. The strategic value of fluorine substitution in drug design is discussed in terms of chem. structure and basic concepts in drug metab. and drug toxicity.
- 23Murphy, C. D.; Sandford, G. Recent advances in fluorination techniques and their anticipated impact on drug metabolism and toxicity. Expert Opin. Drug Metab. Toxicol. 2015, 11, 589– 599, DOI: 10.1517/17425255.2015.1020295[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXksFymt78%253D&md5=b5c542a9e9d7e3c246d4f24da4918f2bRecent advances in fluorination techniques and their anticipated impact on drug metabolism and toxicityMurphy, Cormac D.; Sandford, GrahamExpert Opinion on Drug Metabolism & Toxicology (2015), 11 (4), 589-599CODEN: EODMAP; ISSN:1742-5255. (Informa Healthcare)A review. Introduction: Fluorine's unique physicochem. properties make it a key element for incorporation into pharmacol. active compds. Its presence in a drug can alter a no. of characteristics that affect ADME-Tox, which has prompted efforts at improving synthetic fluorination procedures. Areas covered: This review describes the influence of fluorine on attributes such as potency, lipophilicity, metabolic stability and bioavailability and how the effects obsd. are related to the physicochem. characteristics of the element. Examples of more recently used larger scale synthetic methods for introduction of fluorine into drug leads are detailed and the potential for using biol. systems for fluorinated drug prodn. is discussed. Expert opinion: The synthetic procedures for carbon-fluorine bond formation largely still rely on decades-old technol. for the manufg. scale and new reagents and methods are required to meet the demands for the prepn. of structurally more complex drugs. The improvement of in vitro and computational methods should make fluorinated drug design more efficient and place less emphasis on approaches such as fluorine scanning and animal studies. The introduction of new fluorinated drugs, and in particular those that have novel fluorinated functional groups, should be accompanied by rigorous environmental assessment to det. the nature of transformation products that may cause ecol. damage.
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- 28Nodwell, M. B.; Yang, H.; Colovic, M.; Yuan, Z.; Merkens, H.; Martin, R. E.; Benard, F.; Schaffer, P.; Britton, R. 18)F-Fluorination of unactivated C-H bonds in branched aliphatic amino acids: Direct synthesis of oncological positron emission tomography imaging agents. J. Am. Chem. Soc. 2017, 139, 3595– 3598, DOI: 10.1021/jacs.6b11533[ACS Full Text], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjsVyqtrc%253D&md5=0a17fa3fdaa61c7b3c5e7dda1ea8975818F-Fluorination of unactivated C-H bonds in branched aliphatic amino acids: Direct synthesis of oncological positron emission tomography imaging agentsNodwell, Matthew B.; Yang, Hua; Colovic, Milena; Yuan, Zheliang; Merkens, Helen; Martin, Rainer E.; Benard, Francois; Schaffer, Paul; Britton, RobertJournal of the American Chemical Society (2017), 139 (10), 3595-3598CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A mild and selective photocatalytic C-H 18F-fluorination reaction has been developed that provides direct access to 18F-fluorinated amino acids. The biodistribution and uptake of three 18F-labeled leucine analogs via LAT1 mediated transport in several cancer cell lines is reported. Positron emission tomog. imaging of mice bearing PC3 (prostate) or U87 (glioma) xenografts using 5-[18F]-fluorohomoleucine showed high tumor uptake and excellent tumor visualization, highlighting the utility of this strategy for rapid tracer discovery for oncol.
- 29Limanto, J.; Shafiee, A.; Devine, P. N.; Upadhyay, V.; Desmond, R. A.; Foster, B. R.; Gauthier, D. R., Jr.; Reamer, R. A.; Volante, R. P. An efficient chemoenzymatic approach to (S)-γ-fluoroleucine ethyl ester. J. Org. Chem. 2005, 70, 2372– 2375, DOI: 10.1021/jo047918j[ACS Full Text], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1yisrs%253D&md5=1677d7e61b539f1ad4e19866ac7fdf92An Efficient Chemoenzymatic Approach to (S)-γ-Fluoroleucine Ethyl EsterLimanto, John; Shafiee, Ali; Devine, Paul N.; Upadhyay, Veena; Desmond, Richard A.; Foster, Bruce R.; Gauthier, Donald R., Jr.; Reamer, Robert A.; Volante, R. P.Journal of Organic Chemistry (2005), 70 (6), 2372-2375CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)This work describes the asym. synthesis of (S)-γ-fluoroleucine Et ester (I) from Ph2C:NCH2CO2Et starting material. The key reaction step involves a lipase-catalyzed dynamic ring-opening of 2-(3-butenyl)azlactone II (prepd. from Ph2C:NCH2CO2Et in 5 steps) with EtOH to give N-(pentenoyl)fluoroleucine ester III in 84% enantiomeric excess. Removal of the N-pentenoyl group from III with N,N'-dibromodimethylhydantoin (DBDMH) in the presence of TFA afforded I, which was isolated as its hydrogen sulfate salt in 75% yield and >97% enantiomeric excess.
- 30O’Shea, P. D.; Chen, C. Y.; Gauvreau, D.; Gosselin, F.; Hughes, G.; Nadeau, C.; Volante, R. P. A practical enantioselective synthesis of odanacatib, a potent Cathepsin K inhibitor, via triflate displacement of an α-trifluoromethylbenzyl triflate. J. Org. Chem. 2009, 74, 1605– 1610, DOI: 10.1021/jo8020314[ACS Full Text], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXlt1Sksw%253D%253D&md5=1edffc0a6a028f3e863effbe5a7c9639A Practical Enantioselective Synthesis of Odanacatib, a Potent Cathepsin K Inhibitor, via Triflate Displacement of an α-Trifluoromethylbenzyl TriflateO'Shea, Paul D.; Chen, Cheng-yi; Gauvreau, Danny; Gosselin, Francis; Hughes, Greg; Nadeau, Christian; Volante, Ralph P.Journal of Organic Chemistry (2009), 74 (4), 1605-1610CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)An enantioselective synthesis of the Cathepsin K inhibitor odanacatib (I, MK-0822) is described. The key step involves the novel stereospecific SN2 triflate displacement of a chiral α-trifluoromethylbenzyl triflate II with (S)-γ-fluoroleucine Et ester (III) to generate the required α-trifluoromethylbenzyl amino stereocenter. The triflate displacement is achieved in high yield (95%) and minimal loss of stereochem. The overall synthesis of I is completed in 6 steps in 61% overall yield.
- 31Wang, L.; Wei, J.; Wu, R.; Cheng, G.; Li, X.; Hu, J.; Hu, Y.; Sheng, R. The stability and reactivity of tri-, di-, and monofluoromethyl/methoxy/methylthio groups on arenes under acidic and basic conditions. Org. Chem. Front. 2017, 4, 214– 223, DOI: 10.1039/C6QO00674D[Crossref], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvVKqtbfK&md5=05878f761980875b1f59c72753825a4eThe stability and reactivity of tri-, di-, and monofluoromethyl/methoxy/methylthio groups on arenes under acidic and basic conditionsWang, Lingfei; Wei, Jun; Wu, Ranran; Cheng, Gang; Li, Xinjin; Hu, Jinbo; Hu, Yongzhou; Sheng, RongOrganic Chemistry Frontiers (2017), 4 (2), 214-223CODEN: OCFRA8; ISSN:2052-4129. (Royal Society of Chemistry)In this study, the stability and reactivity of tri-, di- and monofluoromethyl/methoxy/methylthio moiety groups on arenes under acidic or basic conditions was carefully investigated.
- 32Deng, H.; O’Hagan, D.; Schaffrath, C. Fluorometabolite biosynthesis and the fluorinase from Streptomyces cattleya. Nat. Prod. Rep. 2004, 21, 773– 784, DOI: 10.1039/b415087m[Crossref], [PubMed], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXmtVemug%253D%253D&md5=422f9ea144e9a98acd421b4f0df174b4Fluorometabolite biosynthesis and the fluorinase from Streptomyces cattleyaDeng, Hai; O'Hagan, David; Schaffrath, ChristophNatural Product Reports (2004), 21 (6), 773-784CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)A review. This review outlines the recent developments in uncovering the enzymes and intermediates involved in fluorometabolite biosynthesis in the bacterium Streptomyces cattleya. A particular emphasis is placed on the purifn. and characterization of the fluorinase, the C-F bond forming enzyme which initiates the biosynthesis. Nature has hardly developed a biochem. around fluorine, yet fluorinated orgs. are important com. entities, therefore a biotransformation from inorg. to org. fluorine is novel and of contemporary interest.
- 33Lee, S. T.; Cook, D.; Pfister, J. A.; Allen, J. G.; Colegate, S. M.; Riet-Correa, F.; Taylor, C. M. Monofluoroacetate-containing plants that are potentially toxic to livestock. J. Agric. Food Chem. 2014, 62, 7345– 7354, DOI: 10.1021/jf500563h[ACS Full Text], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmtVWhtbY%253D&md5=6368642dc811212d4ee11ac613a6a562Monofluoroacetate-Containing Plants That Are Potentially Toxic to LivestockLee, Stephen T.; Cook, Daniel; Pfister, James A.; Allen, Jeremy G.; Colegate, Steven M.; Riet-Correa, Franklin; Taylor, Charlotte M.Journal of Agricultural and Food Chemistry (2014), 62 (30), 7345-7354CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)A review. Many plants worldwide contain monofluoroacetate and cause sudden death in livestock. These plants are primarily found in the southern continents of Africa, Australia, and South America, where they neg. affect livestock prodn. This review highlights past and current research investigating the plants reported to contain monofluoroacetate and cause sudden death; the mode of action, clin. signs, and pathol. assocd. with poisoning by monofluoroacetate-contg. plants; chem. for the anal. of monofluoroacetate in plants; the coevolution of native flora and fauna in Western Australia with respect to monofluoroacetate-contg. plants; and methods to mitigate livestock losses caused by monofluoroacetate-contg. plants.
- 34Deng, H.; O’Hagan, D. The fluorinase, the chlorinase and the duf-62 enzymes. Curr. Opin. Chem. Biol. 2008, 12, 582– 592, DOI: 10.1016/j.cbpa.2008.06.036[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXht1KgsbbL&md5=b94069dc2015c2fb91f65b1a20c44cd6The fluorinase, the chlorinase and the duf-62 enzymesDeng, Hai; O'Hagan, DavidCurrent Opinion in Chemical Biology (2008), 12 (5), 582-592CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. The fluorinase from Streptomyces cattleya and chlorinase from Salinispora tropica have a commonality in that they mediate nucleophilic reactions of their resp. halide ions to the C-5' carbon of S-adenosyl-L-methionine (SAM). These enzyme reactions fall into the relatively small group of SN2 substitution reactions found in enzymol. These enzymes have some homol. to a larger class of proteins expressed by the duf-62 gene, of which around 200 representatives have been sequenced and deposited in databases. The duf-62 genes express a protein which mediates a hydrolytic cleavage of SAM to generate adenosine and L-methionine. Superficially this enzyme operates very similarly to the halogenases in that water/hydroxide replaces the halide ion. However structural examn. of the duf-62 gene product reveals a very different organization of the active site suggesting a novel mechanism for water activation.
- 35Champagne, P. A.; Pomarole, J.; Therien, M. E.; Benhassine, Y.; Beaulieu, S.; Legault, C. Y.; Paquin, J. F. Enabling nucleophilic substitution reactions of activated alkyl fluorides through hydrogen bonding. Org. Lett. 2013, 15, 2210– 2213, DOI: 10.1021/ol400765a[ACS Full Text], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXms1Wisrw%253D&md5=f3a55eb15516e129876c65a0b13b4061Enabling Nucleophilic Substitution Reactions of Activated Alkyl Fluorides through Hydrogen BondingChampagne, Pier Alexandre; Pomarole, Julien; Therien, Marie-Eve; Benhassine, Yasmine; Beaulieu, Samuel; Legault, Claude Y.; Paquin, Jean-FrancoisOrganic Letters (2013), 15 (9), 2210-2213CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)It was discovered that the presence of water as a cosolvent enables the reaction of activated alkyl fluorides for bimol. nucleophilic substitution reactions. DFT calcns. show that activation proceeds through stabilization of the transition structure by a stronger F···H2O interaction and diminishing C-F bond elongation, and not simple transition state electrostatic stabilization. Overall, the findings put forward a distinct strategy for C-F bond activation through H-bonding.
- 36O’Hagan, D.; Deng, H. Enzymatic fluorination and biotechnological developments of the fluorinase. Chem. Rev. 2015, 115, 634– 649, DOI: 10.1021/cr500209t[ACS Full Text], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1Wlu7zK&md5=316520768c80bde6f16f7248ef8f7734Enzymatic Fluorination and Biotechnological Developments of the FluorinaseO'Hagan, David; Deng, HaiChemical Reviews (Washington, DC, United States) (2015), 115 (2), 634-649CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review focusing on the biosynthesis fluorinated metabolites in nature, the enzymes involeved in fluorination and possible biotech. applications of these enzymes.
- 37Senn, H. M.; O’Hagan, D.; Thiel, W. Insight into enzymatic C–F bond formation from QM and QM/MM calculations. J. Am. Chem. Soc. 2005, 127, 13643– 13655, DOI: 10.1021/ja053875s[ACS Full Text], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXpvFSnurg%253D&md5=158d59340bfea62971bb86cb66d1570fInsight into Enzymatic C-F Bond Formation from QM and QM/MM CalculationsSenn, Hans Martin; O'Hagan, David; Thiel, WalterJournal of the American Chemical Society (2005), 127 (39), 13643-13655CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The C-F bond-forming step in the fluorinase, the only native fluorination enzyme characterized to date, has been studied. The enzyme catalyzes the reaction between S-adenosyl-L-methionine (SAM) and fluoride ions to form 5'-fluoro-5'-deoxyadenosine (5'-FDA) and L-methionine. To obtain an insight into the mechanism of this unusual enzymic reaction and to elucidate the role of the enzyme in catalysis, we have explored the conformational energetics of SAM and the intrinsic reactivity patterns of SAM and fluoride with DFT (BP86) and continuum solvent methods, before investigating the full enzymic system with combined DFT/CHARMM calcns. We find that the enzymic reaction follows an SN2 reaction mechanism, concurring with the intrinsic reactivity preferences in soln. The formation of sulfur ylides is thermodynamically strongly disfavored, and an alternative elimination-addn. mechanism involving the concerted anti-Markovnikov addn. of HF to an enol ether is energetically viable, but kinetically prohibitive. The SN2 activation energy is 92 (112) kJ mol-1 in soln., but only 53 (63) kJ mol-1 in the enzyme, and the reaction energy in the enzyme is -25 (-34) kJ mol-1 (values in parentheses are B3LYP single-point energies). The fluorinase thus lowers the barrier for C-F bond formation by 39 (49) kJ mol-1. A decompn. anal. shows that the major role of the enzyme is in the prepn. and positioning of the substrates.
- 38Zhu, X.; Robinson, D. A.; McEwan, A. R.; O’Hagan, D.; Naismith, J. H. Mechanism of enzymatic fluorination in Streptomyces cattleya. J. Am. Chem. Soc. 2007, 129, 14597– 14604, DOI: 10.1021/ja0731569[ACS Full Text], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1KisbfP&md5=b5155938982c64f203b4d81e1a1dfb9bMechanism of Enzymatic Fluorination in Streptomyces cattleyaZhu, Xiaofeng; Robinson, David A.; McEwan, Andrew R.; O'Hagan, David; Naismith, James H.Journal of the American Chemical Society (2007), 129 (47), 14597-14604CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Recently a fluorination enzyme was identified and isolated from Streptomyces cattleya, as the first committed step on the metabolic pathway to the fluorinated metabolites, fluoroacetate and 4-fluorothreonine. This enzyme, 5'-fluoro-5'-deoxy adenosine synthetase (FDAS), has been shown to catalyze C-F bond formation by nucleophilic attack of fluoride ion to S-adenosyl-L-methionine (SAM) with the concomitant displacement of L-methionine to generate 5'-fluoro-5'-deoxy adenosine (5'-FDA). Although the structures of FDAS bound to both SAM and products have been solved, the mol. mechanism remained to be elucidated. We now report site-directed mutagenesis studies, structural analyses, and isothermal calorimetry (ITC) expts. The data establish the key residues required for catalysis and the order of substrate binding. Fluoride ion is not readily distinguished from water by protein X-ray crystallog.; however, using chloride ion (also a substrate) with a mutant of low activity has enabled the halide ion to be located in nonproductive co-complexes with SAH and SAM. The kinetic data suggest the pos. charged sulfur of SAM is a key requirement in stabilizing the transition state. We propose a mol. mechanism for FDAS in which fluoride weakly assocs. with the enzyme exchanging two water mols. for protein ligation. The binding of SAM expels remaining water assocd. with fluoride ion and traps the ion in a pocket positioned to react with SAM, generating L-methionine and 5'-FDA. L-Methionine then dissocs. from the enzyme followed by 5'-FDA.
- 39Murphy, C. D.; O’Hagan, D.; Schaffrath, C. Identification of a PLP-dependent threonine transaldolase: A novel enzyme involved in 4-fluorothreonine biosynthesis in Streptomyces cattleya. Angew. Chem., Int. Ed. 2001, 40, 4479– 4481, DOI: 10.1002/1521-3773(20011203)40:23<4479::AID-ANIE4479>3.0.CO;2-1[Crossref], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXptlWhtbk%253D&md5=bf7ded2b1b7e78827d49625c641379e9Identification of a PLP-dependent threonine transaldolase: A novel enzyme involved in 4-fluorothreonine biosynthesis in Streptomyces cattleyaMurphy, Cormac D.; O'Hagan, David; Schaffrath, ChristophAngewandte Chemie, International Edition (2001), 40 (23), 4479-4481CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)The final enzyme on the biosynthetic pathway to 4-fluorothreonine is identified in Streptomyces cattleya. The enzyme catalyzes a pyridoxal phosphate-dependent "transaldose" reaction between L-threonine and fluoroacetaldehyde (PLP = pyridoxal 5'-phosphate). Unlike threonine aldolases, glycine is not a substrate for this new type of enzyme.
- 40Xu, X. H.; Yao, G. M.; Li, Y. M.; Lu, J. H.; Lin, C. J.; Wang, X.; Kong, C. H. 5-Fluorouracil derivatives from the sponge Phakellia fusca. J. Nat. Prod. 2003, 66, 285– 288, DOI: 10.1021/np020034f[ACS Full Text], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhtlCltA%253D%253D&md5=9f1432a7c640bd8b57437ed80ae814995-Fluorouracil Derivatives from the Sponge Phakellia fuscaXu, Xiao-Hua; Yao, Guang-Min; Li, Yan-Ming; Lu, Jian-Hua; Lin, Chang-Jiang; Wang, Xin; Kong, Chui-HuaJournal of Natural Products (2003), 66 (2), 285-288CODEN: JNPRDF; ISSN:0163-3864. (American Chemical Society)5-Fluorouracil derivs. were isolated from the marine sponge Phakellia fusca collected around the Yongxing Island of the Xisha Islands in the South Sea of China. Their structures were detd. on the basis of spectral anal. and X-ray diffraction.
- 41Goncharov, N. V.; Jenkins, R. O.; Radilov, A. S. Toxicology of fluoroacetate: a review, with possible directions for therapy research. J. Appl. Toxicol. 2006, 26, 148– 161, DOI: 10.1002/jat.1118[Crossref], [PubMed], [CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjtFerurs%253D&md5=d2e2b695c218a75c93b6a0c779e0bebbToxicology of fluoroacetate: a review, with possible directions for therapy researchGoncharov, Nikolay V.; Jenkins, Richard O.; Radilov, Andrey S.Journal of Applied Toxicology (2006), 26 (2), 148-161CODEN: JJATDK; ISSN:0260-437X. (John Wiley & Sons Ltd.)A review. Fluoroacetate (FA; CH2FCOOR) is highly toxic towards humans and other mammals through inhibition of the enzyme aconitase in the tricarboxylic acid cycle, caused by 'lethal synthesis' of an isomer of fluorocitrate (FC). FA is found in a range of plant species and their ingestion can cause the death of ruminant animals. Some fluorinated compds. used as anticancer agents, narcotic analgesics, pesticides or industrial chems. metabolize to FA as intermediate products. The chem. characteristics of FA and the clin. signs of intoxication warrant the re-evaluation of the toxic danger of FA and renewed efforts in the search for effective therapeutic means. Antidotal therapy for FA intoxication was aimed at preventing fluorocitrate synthesis and aconitase blockade in mitochondria, and at providing citrate outflow from this organelle. Despite a greatly improved understanding of the biochem. mechanism of FA toxicity, ethanol, if taken immediately after the poisoning, was the most acceptable antidote for the past 6 decades. This review deals with the clin. signs and physiol. and biochem. mechanisms of FA intoxication to provide an explanation of why, even after decades of investigation, has no effective therapy to FA intoxication been elaborated. An apparent lack of integrated toxicol. studies is viewed as a limiter of progress in this regard. Two principal ways of developing effective therapies for FA intoxication are considered. Firstly, competitive inhibition of FA interaction with CoA and of FC interaction with aconitase. Secondly, channeling the alternative metabolic pathways by orienting the fate of citrate via cytosolic aconitase, and by maintaining the flux of reducing equiv. into the TCA cycle via glutamate dehydrogenase.
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- 43McCombie, H.; Saunders, B. C. Fluoroacetates and allied compounds. Nature 1946, 158, 382– 385, DOI: 10.1038/158382a0[Crossref], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaH2sXhtlOltg%253D%253D&md5=7c7d3a6b44d34cf35f49ca5d7da5354eFluoroacetates and allied compoundsMcCombie, H.; Saunders, B. C.Nature (London, United Kingdom) (1946), 158 (), 382-5CODEN: NATUAS; ISSN:0028-0836.Convulsant poisons with delayed action contg. the -CH2F group have been synthesized and investigated pharmacologically. The firm binding of the F renders chem. detection and decontamination difficult. A compd. of outstanding interest is 2-fluoroethyl fluoroacetate FCH2COOCH2CH2F (I) prepd. by the action of fluoroacetyl chloride (II) on 2-fluoroethyl alc. (III). A 10-min. exposure to 0.092 g. I/cu. m. killed 70% of the rabbits, guinea pigs, and rats tested and 0.05 g. I/cu. m. killed 50% of rabbits. About half as toxic are Me fluoroacetate (IV), b. 104° m. -35° and Et, Pr, and iso-Pr fluoroacetates, prepd. by heating the corresponding chloroacetates with KF in a rotating autoclave. IV usually produced convulsions 30-60 min. after exposure and death within a few hrs.; 0.1 g./cu. m. inhaled 10 min. killed 50% of the rabbits and guinea pigs (mice were somewhat more resistant), and 0.25 mg./kg. injected intravenously killed 50% of the rabbits. Claisen ester condensation of IV gave Me α γ-difluoroacetoacetate. Fluoroacetamide (V), prepd. from NH3 and IV, was useful for characterization purposes and as an intermediate in the prepn. of fluoroacetic acid (VI). V and VI were as toxic as IV on intravenous injection into rabbits. FCH2CN, b. 80° prepd. by distn. of V with phosphoric anhydride, was more toxic to rabbits than to smaller animals. Substituted amides prepd. included FCH2CONHMe, FCH2CON(NO)Me, FCH2CONH(CH2)2OH, FCH2CONH(CH2)2Cl, and FCH2CON(CH2CH2Cl)2. Na fluoroacetate (VII) was obtained by a new method from IV. II and fluoroacetyl fluoride were similar to IV in toxicity. Fluoroacetic anhydride, prepd. from VII and II, were rather more toxic by inhalation than IV. Et N-fluoroacetyl-glycine and cholesterol fluoroacetate were considerably less toxic than IV. III, b. 101° completely miscible with H2O, prepd. by heating ethylene chlorohydrin with KF in the rotating autoclave, was about as toxic as IV. I, III, and IV had extremely faint odors. Fluoroacetaldehyde, prepd. by oxidation of III with MnO2 and H2SO4, was a liquid which polymerized on standing and was similar to III in toxicity. FCH2CH2Cl obtained, by the action of SOCl2 on III, was nontoxic; it reacted with C6H5ONa to give Ph 2-fluoroethyl ether, considerably less toxic than IV. FCH2CH2Br (VIII) reacted with KSCN to give fluoroethyl thiocyanate, which on treatment with Cl-H2O gave fluoroethylsulfonyl chloride, nontoxic. Bis(2-fluoroethyl) sulfate, (FCH2CH2O)2SO2, proved useful as a fluoroethylating agent, e.g., in the reaction with β-naphthol in alk. soln. to form fluoroethyl naphthyl ether. 2-Chloroethyl fluoroacetate and 2-fluoroethyl chloroacetate (IX) were somewhat more toxic, while 2-fluoroethyl acetate and FCH2COSCH2CH2Cl were less toxic than IV. S,S'-Bis(2-fluoroethyl)-dithioglycol (X) FCH2CH2SCH2CH2SCH2CH2F, ("sesqui-fluoro-H") was nonvesicant and nontoxic, in contrast to S, S'-bis(2-chloroethyl)dithioglycol ("sesqui-H"). X was prepd. by the following reactions: VIII + NaSH → FCH2CH2SH → FCH2CH2SNa (XI); VIII + 2 XI → X. VIII reacted with trimethylamine, triethylamine, and pyridine to form (2-fluoroethyl)trimethylammonium bromide, (2-fluoroethyl)triethylammonium bromide, and (2-fluoroethyl)pyridinium bromide, resp., all not very toxic. Treatment of VIII with dimethylaniline gave 2-Fluoroethylglycine-HCl, prepd. by Fischer-Speier esterification of glycine with III, and 2-fluoroethylbetaine-HCl, prepd. by reaction of anhyd. trimethylamine and IX, had subcutaneous LD50 values of 10 and 45 mg./kg. for mice. Fluoroacetylsalicyclic acid (fluoroaspirin) caused initial stupor without convulsions in mice. Bis(2-fluoroethyl)fluorophosphonate, obtained by the action of POCl2F on III caused miosis, and at a concn. of 0.5 g./cu. m. (10 min. exposure) produced in 2 of 6 rats a remarkable state of hyperactivity followed by convulsions, coma, and death. Triethyllead fluoroacetate, FCH2COOPbEt3, had sternutatory and, upon injection, convulsant properties. No toxic action was shown by compds. devoid of the FCH2- group, i.e. Me α-fluoropropionate, Me α-fluoroisobutyrate, chloroacetyl fluoride, and Et fluoroformate.
- 44Lauble, H.; Kennedy, M. C.; Emptage, M. H.; Beinert, H.; Stout, C. D. The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 13699– 13703, DOI: 10.1073/pnas.93.24.13699[Crossref], [PubMed], [CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xnt1Gnsbo%253D&md5=ed8ef16ac9d474a434754199da264f02The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complexLauble, H.; Kennedy, M. C.; Emptage, M. H.; Beinert, H.; Stout, C. D.Proceedings of the National Academy of Sciences of the United States of America (1996), 93 (24), 13699-13703CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)It has been known for many years that fluoroacetate and fluorocitrate when metabolized are highly toxic, and that at least one effect of fluorocitrate is to inactivate aconitase. Here, the authors present evidence supporting the hypothesis that the (-)-erythro diastereomer of 2-fluorocitrate acts as a mechanism-based inhibitor of aconitase by 1st being converted to fluoro-cis-aconitate, followed by the addn. of hydroxide and with loss of fluoride to form 4-hydroxy-trans-aconitate (HTn), which binds very tightly, but not covalently, to the enzyme. The formation of HTn by these reactions is in accord with the working model for the enzyme mechanism. That HTn is the product of fluorocitrate inhibition is supported by the crystal structure of the enzyme-inhibitor complex at 2.05-Å resoln., release of fluoride stoichiometric with total enzyme when (-)-erythro-2-fluorocitrate is added, HPLC anal. of the product, slow displacement of HTn by 106-fold excess of isocitrate, and previously published Moessbauer expts. When (+)-erythro-2-fluorocitrate is added to aconitase, the release of fluoride is stoichiometric with total substrate added, and HPLC anal. of the products indicates the formation of oxalosuccinate, and its deriv., α-ketoglutarate. This is consistent with the proposed mechanism, as is the formation of HTn from (-)-erythro-2-fluorocitrate. The structure of the inhibited complex reveals that HTn binds like the inhibitor, trans-aconitate, while providing all the interactions of the natural substrate, isocitrate. The structure exhibits 4 H-bonds <2.7 Å in length involving HTn, H2O bound to the [4Fe-4S] cluster, Asp-165, and His-167, as well as low temp. factors for these moieties, consistent with the obsd. very tight binding of the inhibitor.
- 45Hamel, J. D.; Paquin, J. F. Activation of C–F bonds α to C–C multiple bonds. Chem. Commun. (Cambridge, U. K.) 2018, 54, 10224– 10239, DOI: 10.1039/C8CC05108A[Crossref], [PubMed], [CAS], Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsFCktbfN&md5=4c6e994215ac89974c861999f74f7b80Activation of C-F bonds α to C-C multiple bondsHamel, Jean-Denys; Paquin, Jean-FrancoisChemical Communications (Cambridge, United Kingdom) (2018), 54 (73), 10224-10239CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A review. C-F bond activation has drawn a lot of attention in the past, and the case of C(sp3)-F bonds is particularly interesting as those are the strongest single bonds carbon makes with any other element. This Feature Article aims at highlighting the authors' work and that of others on the successful approaches that led to the activation of such C-F bonds at benzylic, allylic, propargylic and allenylic positions, for both mono- and polyfluorides. The authors now hope that this will set ground for discussions, contribute to fostering new ideas, and thus help in pushing current frontiers further.
- 46Sherley, M. The traditional categories of fluoroacetate poisoning signs and symptoms belie substantial underlying similarities. Toxicol. Lett. 2004, 151, 399– 406, DOI: 10.1016/j.toxlet.2004.03.013[Crossref], [PubMed], [CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXlslyitLw%253D&md5=17bea1d92ea6abe9db66e73d61e04d97The traditional categories of fluoroacetate poisoning signs and symptoms belie substantial underlying similaritiesSherley, MirandaToxicology Letters (2004), 151 (3), 399-406CODEN: TOLED5; ISSN:0378-4274. (Elsevier Ireland Ltd.)A review. Sodium monofluoroacetate (Compd. 1080) has been widely used around the world as a vertebrate pest control agent. Following ingestion of 1080 there is a latent period, during which the compd. is metabolized into a toxic form, before the onset of symptoms. The timing of this period varies significantly between species as does the median LD. Traditionally different species have also been classified into groups depending on the primary organ system involved in 1080 toxicosis (cardiac, nervous, or mixed signs/symptoms). However, general acceptance of this method of classification has obscured the fact that several signs of fluoroacetate poisoning are common to most vertebrate species. This paper reviews 5 decades of literature on the signs/symptoms of fluoroacetate poisoning in vertebrates and concludes that there is little justification for the division of animals poisoned by fluoroacetate into symptomatic groups.
- 47Proudfoot, A. T.; Bradberry, S. M.; Vale, J. A. Sodium fluoroacetate poisoning. Toxicol. Rev. 2006, 25, 213– 219, DOI: 10.2165/00139709-200625040-00002[Crossref], [PubMed], [CAS], Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjs1Gksrs%253D&md5=7229d74004657a0ba412497dc809e2edSodium fluoroacetate poisoningProudfoot, Alex T.; Bradberry, Sally M.; Vale, J. AllisterToxicological Reviews (2006), 25 (4), 213-219CODEN: TROEB5; ISSN:1176-2551. (Adis International Ltd.)A review. Sodium fluoroacetate was introduced as a rodenticide in the US in 1946. However, its considerable efficacy against target species is offset by comparable toxicity to other mammals and, to a lesser extent, birds and its use as a general rodenticide was therefore severely curtailed by 1990. Currently, sodium fluoroacetate is licensed in the US for use against coyotes, which prey on sheep and goats, and in Australia and New Zealand to kill unwanted introduced species. The extreme toxicity of fluoroacetate to mammals and insects stems from its similarity to acetate, which has a pivotal role in cellular metab. Fluoroacetate combines with CoA (CoA-SH) to form fluoroacetyl CoA, which can substitute for acetyl CoA in the tricarboxylic acid cycle and reacts with citrate synthase to produce fluorocitrate, a metabolite of which then binds very tightly to aconitase thereby halting the cycle. Many of the features of fluoroacetate poisoning are, therefore, largely direct and indirect consequences of impaired oxidative metab. Energy prodn. is reduced and intermediates of the tricarboxylic acid cycle subsequent to citrate are depleted. Among these is oxoglutarate, a precursor of glutamate, which is not only an excitatory neurotransmitter in the CNS but is also required for efficient removal of ammonia via the urea cycle. Increased ammonia concns. may contribute to the incidence of seizures. Glutamate is also required for glutamine synthesis and glutamine depletion has been obsd. in the brain of fluoroacetate-poisoned rodents. Reduced cellular oxidative metab. contributes to a lactic acidosis. Inability to oxidize fatty acids via the tricarboxylic acid cycle leads to ketone body accumulation and worsening acidosis. ATP (ATP) depletion results in inhibition of high energy-consuming reactions such as gluconeogenesis. Fluoroacetate poisoning is assocd. with citrate accumulation in several tissues, including the brain. Fluoride liberated from fluoroacetate, citrate and fluorocitrate are calcium chelators and there are both animal and clin. data to support hypocalcemia as a mechanism of fluoroacetate toxicity. However, the available evidence suggests the fluoride component does not contribute. Acute poisoning with sodium fluoroacetate is uncommon. Ingestion is the major route by which poisoning occurs. Nausea, vomiting and abdominal pain are common within 1 h of ingestion. Sweating, apprehension, confusion and agitation follow. Both supraventricular and ventricular arrhythmias have been reported and nonspecific ST- and T-wave changes are common, the QTc may be prolonged and hypotension may develop. Seizures are the main neurol. feature. Coma may persist for several days. Although several possible antidotes have been investigated, they are of unproven value in humans. The immediate, and probably only, management of fluoroacetate poisoning is therefore supportive, including the correction of hypocalcemia.
- 48Kirsten, E.; Sharma, M. L.; Kun, E. Molecular toxicology of (−)-erythro-fluorocitrate: selective inhibition of citrate transport in mitochondria and the binding of fluorocitrate to mitochondrial proteins. Mol. Pharmacol. 1978, 14, 172– 184[PubMed], [CAS], Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXhsVKqs7c%253D&md5=a99fce7d622718212edbb608d9c716deMolecular toxicology of (-)-erythro-fluorocitrate: selective inhibition of citrate transport in mitochondria and the binding of fluorocitrate to mitochondrial proteinsKirsten, Eva; Sharma, Manohar L.; Kun, ErnestMolecular Pharmacology (1978), 14 (1), 172-84CODEN: MOPMA3; ISSN:0026-895X.The rate of entry of oxidizable carboxylic acids into the mitoplast compartment of lysosome- and microsome-free mitochondria was detd. by monitoring substrate-dependent incorporation of 32P-labeled orthophosphate into ADP to form ATP [56-65-5]. The maximal rate of ATP synthesis that was dependent on tricarboxylic acids required the presence of (-)-erythro-fluoromalate [34917-27-6], replacing the physiol. activator L-malate. Previous incubation of mitochondria for 5-15 min (at 25-30°) with 50 pmols of (-)-erythro-fluorocitrate [34917-26-5]/mg mitochondrial protein irreversibly and selectively inhibited citrate [77-92-9] supported ATP synthesis. Previous incubation of mitochondria for 15 min with quantities of (-)-erythro-fluorocitrate that inhibited citrate-dependent ATP synthesis also inhibited mitochondrial citrate-dependent fatty acid synthesis. The utilization of added citrate for fatty acid biosynthesis by the cytoplasmic system was not affected by fluorocitrate. Incubation of inner membrane vesicles with 3,4,5,6-14C-labeled (-)-erythro-fluorocitrate resulted in the covalent binding of fluorocitrate to protein components of the membrane. The bond between fluorocitric acid and protein was specifically cleaved by incubation with 0.4 M neutral hydroxylamine, but not by 1 mM o-phenanthroline. The hydroxamic acid of fluorocitrate, which was formed by treatment of protein-bound fluorocitrate with hydroxylamine, was detected by high-voltage electrophoresis and thin-layer chromatog. Binding of fluorocitrate to inner membrane vesicles dissolved in 4 M guanidine-HCl was prevented by previous incubation of membrane proteins with o-{[3-(hydroxymercuri)-2-methoxypropyl]carbamoyl}phenoxyacetic acid Na salt. The covalent binding of (-)-erythro-fluorocitrate to membrane proteins was the result of an enzymic process that was activated by Mn2+. Oxidizable carboxylic acids did not form covalently bound adducts with inner membrane proteins. It was concluded that (-)-erythro-fluorocitrate specifically inhibited citrate transport by covalent binding to 2 protein fractions assocd. with the mitoplast of liver, kidney, heart, and brain tissue.
- 49Menon, K. I.; Feldwick, M. G.; Noakes, P. S.; Mead, R. J. The mode of toxic action of the pesticide gliftor: the metabolism of 1,3-difluoroacetone to (−)-erythro-fluorocitrate. J. Biochem. Mol. Toxicol. 2001, 15, 47– 54, DOI: 10.1002/1099-0461(2001)15:1<47::AID-JBT6>3.0.CO;2-E[Crossref], [PubMed], [CAS], Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXotFWltA%253D%253D&md5=f768c20bacfd23f6a5cfdec35db822d0The mode of toxic action of the pesticide Gliftor: the metabolism of 1,3-difluoroacetone to (-)-erythro-fluorocitrateMenon, K. I.; Feldwick, M. G.; Noakes, P. S.; Mead, R. J.Journal of Biochemical and Molecular Toxicology (2001), 15 (1), 47-54CODEN: JBMTFQ; ISSN:1095-6670. (John Wiley & Sons, Inc.)The biochem. toxicol. of 1,3-difluoroacetone, a known metabolite of the major ingredient of the pesticide Gliftor (1,3-difluoro-2-propanol), was investigated in vivo and in vitro. Rat kidney homogenates supplemented with CoA, ATP, oxaloacetate, and Mg2+ converted 1,3-difluoroacetone to (-)-erythro-fluorocitrate in vitro. Administration of 1,3-difluoroacetone (100 mg kg-1 body wt.) to rats in vivo resulted in (-)-erythro-fluorocitrate synthesis in the kidney, which was preceded by an elevation in fluoride levels and followed by citrate accumulation. Animals dosed with 1,3-difluoroacetone did not display the 2-3 h lag phase in either (-)-erythro-fluorocitrate synthesis or in citrate and fluoride accumulation characteristic of animals dosed with 1,3-difluoro-2-propanol. We demonstrate that the conversion of 1,3-difluoro-2-propanol to 1,3-difluoroacetone by an NAD+-dependent oxidn. is the rate-limiting step in the synthesis of the toxic product, (-)-erythro-fluorocitrate from 1,3-difluoro-2-propanol. Prior administration of 4-methylpyrazole (90 mg kg-1 body wt.) was shown to prevent the conversion of 1,3-difluoro-2-propanol (100 mg kg-1 body wt.) to (-)-erythro-fluorocitrate in vivo and to eliminate the fluoride and citrate elevations seen in 1,3-difluoro-2-propanol-intoxicated animals. However, administration of 4-methylpyrazole (90 mg kg-1 body wt.) to rats 2 h prior to 1,3-difluoroacetone (100 mg kg-1 body wt.) was ineffective in preventing (-)-erythro-fluorocitrate synthesis and did not diminish fluoride or citrate accumulation in vivo. We conclude that the prophylactic and antidotal properties of 4-methylpyrazole seen in animals treated with 1,3-difluoro-2-propanol derive from its capacity to inhibit the NAD+-dependent oxidn. responsible for converting 1,3-difluoro-2-propanol to 1,3-difluoroacetone in the committed step of the toxic pathway.
- 50Feldwick, M. G.; Noakes, P. S.; Prause, U.; Mead, R. J.; Kostyniak, P. J. The biochemical toxicology of 1,3-difluoro-2-propanol, the major ingredient of the pesticide gliftor: the potential of 4-methylpyrazole as an antidote. J. Biochem. Mol. Toxicol. 1998, 12, 41– 52, DOI: 10.1002/(SICI)1099-0461(1998)12:1<41::AID-JBT6>3.0.CO;2-P[Crossref], [PubMed], [CAS], Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXnsl2ktLc%253D&md5=f87b7ff30d51b11fe12ebf72c023e82fThe biochemical toxicology of 1,3-difluoro-2-propanol, the major ingredient of the pesticide gliftor: the potential of 4-methylpyrazole as an antidoteFeldwick, M. G.; Noakes, P. S.; Prause, U.; Mead, R. J.; Kostyniak, P. J.Journal of Biochemical and Molecular Toxicology (1998), 12 (1), 41-52CODEN: JBMTFQ; ISSN:1095-6670. (John Wiley & Sons, Inc.)Administration to rats of 1,3-difluoro-2-propanol (100 mg kg-1 body wt.), the major ingredient of the pesticide gliftor, resulted in accumulation of citrate in the kidney after a 3 h lag phase. 1,3-Difluoro-2-propanol was found to be metabolized to 1,3-difluoroacetone and ultimately to the aconitate hydratase inhibitor (-)-erythrofluorocitrate and free fluoride. The conversion of 1,3-difluoro-2-propanol to 1,3-difluoroacetone was found to be catalyzed by an NAD+-dependent alc. dehydrogenase, while the defluorination was attributed to microsomal monooxygenase activity induced by phenobarbitone and inhibited by piperonyl butoxide. 4-Methylpyrazole was found to inhibit both of these processes in vitro and when administered (90 mg kg-1 body wt.) to rats, 2 h prior to 1,3-difluoro-2-propanol, eliminated signs of poisoning, prevented (-)-erythrofluorocitrate synthesis, and markedly decreased citrate and fluoride accumulation in vivo. 4-Methylpyrazole also appeared to diminish (-)-erythrofluorocitrate synthesis from fluoroacetate in vivo, and this was attributed to its capacity to inhibit malate dehydrogenase activity. The antidotal potential of 4-methylpyrazole and the potential for 1,3-difluoro-2-propanol to replace fluoroacetate (compd. 1080) as a vertebrate pesticide is discussed.
- 51Leisch, H.; Morley, K.; Lau, P. C. Baeyer-Villiger monooxygenases: more than just green chemistry. Chem. Rev. 2011, 111, 4165– 4222, DOI: 10.1021/cr1003437[ACS Full Text], [CAS], Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXls1elurw%253D&md5=4414d1675ebfeb634c52db50d1625a3dBaeyer-Villiger Monooxygenases: More Than Just Green ChemistryLeisch, Hannes; Morley, Krista; Lau, Peter C. K.Chemical Reviews (Washington, DC, United States) (2011), 111 (7), 4165-4222CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)This review focuses on the monooxygenase-catalyzed Baeyer-Villiger oxidn. of linear or cyclic ketones as a green chem. tool to address environmental sustainability, a system to study its mol. diversity and catalytic mechanism, industrial scale bioprocess development, and protein engineering to evolve new biotechnol. applications.
- 52Fiorentini, F.; Romero, E.; Fraaije, M. W.; Faber, K.; Hall, M.; Mattevi, A. Baeyer-Villiger monooxygenase FMO5 as entry point in drug metabolism. ACS Chem. Biol. 2017, 12, 2379– 2387, DOI: 10.1021/acschembio.7b00470[ACS Full Text], [CAS], Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1yisrfL&md5=f8c5f7d9f23f28fdda7e5de91bcb5ef2Baeyer-Villiger Monooxygenase FMO5 as Entry Point in Drug MetabolismFiorentini, Filippo; Romero, Elvira; Fraaije, Marco W.; Faber, Kurt; Hall, Melanie; Mattevi, AndreaACS Chemical Biology (2017), 12 (9), 2379-2387CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Flavin-contg. monooxygenases (FMOs) are emerging as effective players in oxidative drug metab. Until recently, the functions of the five human FMO isoforms were mostly linked to their capability of oxygenating mols. contg. soft N- and S-nucleophiles. However, the human FMO isoform 5 was recently shown to feature an atypical activity as Baeyer-Villiger monooxygenase. With the aim of evaluating such an alternative entry point in the metab. of active pharmaceutical ingredients, the authors selected and tested drug mols. bearing a carbonyl group on an aliph. chain. Nabumetone and pentoxifylline, two widely used pharmaceuticals, were thereby demonstrated to be efficiently oxidized in vitro by FMO5 to the corresponding acetate esters with high selectivity. The proposed pathways explain the formation of a predominant plasma metabolite of pentoxifylline as well as the crucial transformation of the pro-drug nabumetone into the pharmacol. active compd. Using the recombinant enzyme, the ester derivs. of both drugs were obtained in milligram amts., purified, and fully characterized. This protocol can potentially be extended to other FMO5 candidate substrates as it represents an effective and robust bench-ready platform applicable to API screening and metabolite synthesis.
- 53Fiorentini, F.; Geier, M.; Binda, C.; Winkler, M.; Faber, K.; Hall, M.; Mattevi, A. Biocatalytic characterization of human FMO5: Unearthing Baeyer-Villiger reactions in humans. ACS Chem. Biol. 2016, 11, 1039– 1048, DOI: 10.1021/acschembio.5b01016[ACS Full Text], [CAS], Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XoslymtA%253D%253D&md5=2d328642497c76a7c6789b0e70f1ed7fBiocatalytic Characterization of Human FMO5: Unearthing Baeyer-Villiger Reactions in HumansFiorentini, Filippo; Geier, Martina; Binda, Claudia; Winkler, Margit; Faber, Kurt; Hall, Melanie; Mattevi, AndreaACS Chemical Biology (2016), 11 (4), 1039-1048CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Flavin-contg. mono-oxygenases are known as potent drug-metabolizing enzymes, providing complementary functions to the well-investigated cytochrome P 450 mono-oxygenases. While human FMO isoforms are typically involved in the oxidn. of soft nucleophiles, the biocatalytic activity of human FMO5 (along its physiol. role) has long remained unexplored. In this study, we demonstrate the atypical in vitro activity of human FMO5 as a Baeyer-Villiger mono-oxygenase on a broad range of substrates, revealing the first example to date of a human protein catalyzing such reactions. The isolated and purified protein was active on diverse carbonyl compds., whereas soft nucleophiles were mostly non- or poorly reactive. The absence of the typical characteristic sequence motifs sets human FMO5 apart from all characterized Baeyer-Villiger mono-oxygenases so far. These findings open new perspectives in human oxidative metab.
- 54Kitazume, T.; Kataoka, J. Study on the effect of di- and trifluoromethyl groups on the Baeyer-Villiger reaction. J. Fluorine Chem. 1996, 80, 157– 158, DOI: 10.1016/S0022-1139(96)03500-2[Crossref], [CAS], Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXitVCgsA%253D%253D&md5=1f163743c0fa284d619a6e788312f67fStudy on the effect of di- and trifluoromethyl groups on the Baeyer-Villiger reactionKitazume, Tomoya; Kataoka, JunichiJournal of Fluorine Chemistry (1996), 80 (2), 157-158CODEN: JFLCAR; ISSN:0022-1139. (Elsevier)Case studies of the Baeyer-Villiger reaction applied to di- and trifluoromethyl ketone derivs. and the effect of the fluoromethyl groups on the Baeyer-Villiger reaction, are described.
- 55Kobayashi, S.; Tanaka, H.; Amii, H.; Uneyama, K. A new finding in selective Baeyer-Villiger oxidation of α-fluorinated ketones; a new and practical route for the synthesis of α-fluorinated esters. Tetrahedron 2003, 59, 1547– 1552, DOI: 10.1016/S0040-4020(03)00047-4[Crossref], [CAS], Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhtFCkt70%253D&md5=59ba46de3d9a7cdccdb389013f7bbe45A new finding in selective Baeyer-Villiger oxidation of α-fluorinated ketones; a new and practical route for the synthesis of α-fluorinated estersKobayashi, Satoru; Tanaka, Hiroaki; Amii, Hideki; Uneyama, KenjiTetrahedron (2003), 59 (9), 1547-1552CODEN: TETRAB; ISSN:0040-4020. (Elsevier Science Ltd.)α-Fluorinated esters were effectively prepd. by the Baeyer-Villiger oxidn. of α-fluorinated ketones with m-chloroperbenzoic acid (m-CPBA) under mild conditions. The yield of the esters was influenced by the choice of solvent, base, and substituent on the aryl group of the ketones. 4-Methoxyphenyl substituted fluoro-ketones were oxidized almost quant. with m-CPBA within 10 min to 12 h at room temp. using 1,1,1,3,3,3-hexafluoro-2-propanol as a cosolvent with CH2Cl2 (1:1, vol./vol.) and aq. buffer (KH2PO4-NaOH, pH 7.6) as an additive base. The oxidn. reaction rates of α-fluorinated ketones were higher than those of the corresponding non-fluorinated ketones. The fluorine atom at α-position of fluoromethyl aryl ketones enhanced the reactivity in the Baeyer-Villiger oxidn.
- 56Fischer, R. T.; Trzaskos, J. M.; Magolda, R. L.; Ko, S. S.; Brosz, C. S.; Larsen, B. Lanosterol 14α-methyl demethylase. Isolation and characterization of the third metabolically generated oxidative demethylation intermediate. J. Biol. Chem. 1991, 266, 6124– 6132[PubMed], [CAS], Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXktVequr0%253D&md5=9ffe09b0aa9c75e31bc57062ee2120e8Lanosterol 14α-methyl demethylase. Isolation and characterization of the third metabolically generated oxidative demethylation intermediateFischer, Robert T.; Trzaskos, James M.; Magolda, Ronald L.; Ko, Soo S.; Brosz, Christian S.; Larsen, BarbaraJournal of Biological Chemistry (1991), 266 (10), 6124-32CODEN: JBCHA3; ISSN:0021-9258.Conditions were identified which permit metabolic formation of the 3rd oxidized intermediate in the lanosterol 14α-Me demethylase reaction cascade. Metab. of either the immediate precursor substrate, 3β-hydroxylanost-8-en-32-al, or lanost-8-ene-3β,32-diol under mixed function oxidase conditions afforded formation of the intermediate. It must be emphasized that the intermediate could only be detected if sapon. procedures were omitted during sterol isolation. Comparative chem. and biochem. studies of the isolated metabolite with 3β,15α-dihydroxylanost-8-en-32-al revealed that the metabolite was not the 15α-hydroxylanosterol aldehyde, a putative demethylase intermediate. The metabolite was efficiently converted to the demethylated Δ8,14-diene sterol in the absence of O2 or NADPH, thus supporting its identity as the final oxidized intermediate in the lanosterol 14α-Me demethylase cascade. 1H NMR anal. showed a proton resonance at 7.86 ppm consistent with a formyloxy proton. Mass spectral and IR anal. of the metabolite clearly established O insertion into the immediate precursor substrate, 3β-hydroxylanost-8-en-32-al. Collectively, the biochem. and chem. characteristics of the metabolite supported a structural assignment for the metabolite as 14α-formyloxylanost-8-en-3β-ol.
- 57Vaz, A. D.; Pernecky, S. J.; Raner, G. M.; Coon, M. J. Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 4644– 4648, DOI: 10.1073/pnas.93.10.4644[Crossref], [PubMed], [CAS], Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XjtVKlsr8%253D&md5=1b5010f98101cdc729d53f277671f10ePeroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4Vaz, Alfin D. N.; Pernecky, Steven J.; Raner, Gregory M.; Coon, Minor J.Proceedings of the National Academy of Sciences of the United States of America (1996), 93 (10), 4644-4648CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Among biol. catalysts, cytochrome P 450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chem. reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several labs. points to a hypervalent iron-oxenoid species in P 450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P 450 aromatase and iron-peroxo species is involved. We have shown more recently that purified liver microsomal P 450 cytochromes, including phenobarbital-induced P 450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P 450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (Δ2-27)] makes use of evidence from other labs. that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidn., hydrogen peroxide prodn., and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidn., cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were detd. with P450s 2B4, 2B4 (Δ2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenyl-ethanol (2-fold). In sharp contrast, the deformylation of cyclohexane by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H2O2, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P 450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.
- 58Isin, E. M.; Guengerich, F. P. Complex reactions catalyzed by cytochrome P450 enzymes. Biochim. Biophys. Acta, Gen. Subj. 2007, 1770, 314– 329, DOI: 10.1016/j.bbagen.2006.07.003[Crossref], [PubMed], [CAS], Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXpvVyqsQ%253D%253D&md5=05b988e6536ea1e8725799d3cf395005Complex reactions catalyzed by cytochrome P 450 enzymesIsin, Emre M.; Guengerich, F. PeterBiochimica et Biophysica Acta, General Subjects (2007), 1770 (3), 314-329CODEN: BBGSB3; ISSN:0304-4165. (Elsevier Ltd.)A review. Cytochrome P 450 (P 450) isoforms are some of the most versatile redox proteins known. The basic P 450 reactions include C-hydroxylation, heteroatom oxygenation, heteroatom release (dealkylation), and epoxide formation. Mechanistic explanations for these reactions have been advanced. A no. of more complex P 450 reactions also occur, and these can be understood largely in the context of the basic chem. mechanisms and subsequent rearrangements. The list discussed here updates a 2001 review and includes Cl oxygenation, arom. dehalogenation, formation of diindole products, dimer formation via Diels-Alder reactions of products, ring coupling and also ring formation, reductive activation (e.g., aristolochic acid), ring contraction (piperidine nitroxide radical), oxidn. of troglitazone, cleavage of amino oxazoles and a 1,2,4-oxadiazole ring, bioactivation of a dihydrobenzoxathiin, and oxidative aryl migration.
- 59Guengerich, F. P.; Yoshimoto, F. K. Formation and cleavage of C–C bonds by enzymatic oxidation-reduction reactions. Chem. Rev. 2018, 118, 6573– 6655, DOI: 10.1021/acs.chemrev.8b00031[ACS Full Text], [CAS], Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFOru7fJ&md5=53975521b83d7596b867cf27adbd59b7Formation and Cleavage of C-C Bonds by Enzymatic Oxidation-Reduction ReactionsGuengerich, F. Peter; Yoshimoto, Francis K.Chemical Reviews (Washington, DC, United States) (2018), 118 (14), 6573-6655CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Many oxidn.-redn. (redox) enzymes, particularly oxygenases, have roles in reactions that make and break C-C bonds. The list includes cytochrome P 450 and other heme-based monooxygenases, heme-based dioxygenases, non-heme iron mono- and dioxygenases, flavoproteins, radical S-adenosylmethionine enzymes, copper enzymes, and peroxidases. Reactions involve steroids, intermediary metab., secondary natural products, drugs, and industrial and agricultural chems. Many C-C bonds are formed via either (i) coupling of diradicals or (ii) generation of unstable products that rearrange. C-C cleavage reactions involve several themes: (i) rearrangement of unstable oxidized products produced by the enzymes, (ii) oxidn. and collapse of radicals or cations via rearrangement, (iii) oxygenation to yield products that are readily hydrolyzed by other enzymes, and (iv) activation of O2 in systems in which the binding of a substrate facilitates O2 activation. Many of the enzymes involve metals, but of these iron is clearly predominant.
- 60Cox, C. D.; Coleman, P. J.; Breslin, M. J.; Whitman, D. B.; Garbaccio, R. M.; Fraley, M. E.; Buser, C. A.; Walsh, E. S.; Hamilton, K.; Schaber, M. D.; Lobell, R. B.; Tao, W.; Davide, J. P.; Diehl, R. E.; Abrams, M. T.; South, V. J.; Huber, H. E.; Torrent, M.; Prueksaritanont, T.; Li, C.; Slaughter, D. E.; Mahan, E.; Fernandez-Metzler, C.; Yan, Y.; Kuo, L. C.; Kohl, N. E.; Hartman, G. D. Kinesin spindle protein (KSP) inhibitors. 9. Discovery of (2S)-4-(2,5-difluorophenyl)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide (MK-0731) for the treatment of taxane-refractory cancer. J. Med. Chem. 2008, 51, 4239– 4252, DOI: 10.1021/jm800386y[ACS Full Text], [CAS], Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnsl2rs7s%253D&md5=283d7227309cbe634f18a640c8085090Kinesin Spindle Protein (KSP) Inhibitors. 9. Discovery of (2S)-4-(2,5-Difluorophenyl)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide (MK-0731) for the Treatment of Taxane-Refractory CancerCox, Christopher D.; Coleman, Paul J.; Breslin, Michael J.; Whitman, David B.; Garbaccio, Robert M.; Fraley, Mark E.; Buser, Carolyn A.; Walsh, Eileen S.; Hamilton, Kelly; Schaber, Michael D.; Lobell, Robert B.; Tao, Weikang; Davide, Joseph P.; Diehl, Ronald E.; Abrams, Marc T.; South, Vicki J.; Huber, Hans E.; Torrent, Maricel; Prueksaritanont, Thomayant; Li, Chunze; Slaughter, Donald E.; Mahan, Elizabeth; Fernandez-Metzler, Carmen; Yan, Youwei; Kuo, Lawrence C.; Kohl, Nancy E.; Hartman, George D.Journal of Medicinal Chemistry (2008), 51 (14), 4239-4252CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Inhibition of kinesin spindle protein (KSP) is a novel mechanism for treatment of cancer with the potential to overcome limitations assocd. with currently employed cytotoxic agents. Herein, we describe a C2-hydroxymethyl dihydropyrrole KSP inhibitor (11) that circumvents hERG channel binding and poor in vivo potency, issues that limited earlier compds. from our program. However, introduction of the C2-hydroxymethyl group caused 11 to be a substrate for cellular efflux by P-glycoprotein (Pgp). Utilizing knowledge garnered from previous KSP inhibitors, we found that β-fluorination modulated the pKa of the piperidine nitrogen and reduced Pgp efflux, but the resulting compd. (14) generated a toxic metabolite in vivo. Incorporation of fluorine in a strategic, metabolically benign position by synthesis of an N-methyl-3-fluoro-4-(aminomethyl)piperidine urea led to compd. 30 that has an optimal in vitro and metabolic profile. Compd. 30 (MK-0731) was recently studied in a phase I clin. trial in patients with taxane-refractory solid tumors.
- 61Eichhold, T. H.; Hookfin, E. B.; Taiwo, Y. O.; De, B.; Wehmeyer, K. R. Isolation and quantification of fluoroacetate in rat tissues, following dosing of Z-Phe-Ala-CH2-F, a peptidyl fluoromethyl ketone protease inhibitor. J. Pharm. Biomed. Anal. 1997, 16, 459– 467, DOI: 10.1016/S0731-7085(97)00102-7[Crossref], [PubMed], [CAS], Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXnvFWqsL8%253D&md5=ea69dcdcaff325ed19d40e94c6f96760Isolation and quantification of fluoroacetate in rat tissues, following dosing of Z-Phe-Ala-CH2-F, a peptidyl fluoromethyl ketone protease inhibitorEichhold, Thomas H.; Hookfin, Erin B.; Taiwo, Yetunde O.; De, Biswanath; Wehmeyer, Kenneth R.Journal of Pharmaceutical and Biomedical Analysis (1997), 16 (3), 459-467CODEN: JPBADA; ISSN:0731-7085. (Elsevier Science B.V.)Peptidyl fluoromethyl ketones (PFMK) are irreversible inhibitors of cathepsin B, a cysteine proteinase thought to be involved in the degrdn. of cartilage. It has been speculated that PFMK inhibitors may metabolize in rodents to form fluoroacetate (FAC), an extremely toxic poison. A highly selective and sensitive sepn. and detection scheme was developed to measure trace levels of FAC in rat tissues following PFMK dosing. The procedure consisted of extg. FAC from tissue and spiking the ext. with [18O]2-fluoroacetate (18O-FAC) as an internal std. FAC and 18O-FAC were further isolated from matrix components using ion-exchange, solid-phase extn. The pentafluorobenzyl esters of FAC and 18O-FAC were formed to facilitate the chromatog. sepn. Two-dimensional gas chromatog. coupled with selected-ion-monitoring detection provided the final measurement. The assay had a limit of detection of 2 ng FAC per g tissue, and was capable of accurately quantitating as little as 10 ng FAC per g tissue with a S/N ratio of 40:1. Linearity was established over two orders of magnitude, from 2-500 ng ml-1, with 5 μl injected on-column. The method was used to demonstrate that FAC was formed in rats following dosing with Z-Phe-Ala-CH2-F, a PFMK cathepsin enzyme inhibitor.
- 62Schlichting, I.; Berendzen, J.; Chu, K.; Stock, A. M.; Maves, S. A.; Benson, D. E.; Sweet, R. M.; Ringe, D.; Petsko, G. A.; Sligar, S. G. The catalytic pathway of cytochrome p450cam at atomic resolution. Science 2000, 287, 1615– 1622, DOI: 10.1126/science.287.5458.1615[Crossref], [PubMed], [CAS], Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXhs1Ortbw%253D&md5=468f33a36d3faf0f8ae2476b3e3a640bThe catalytic pathway of cytochrome P450cam at atomic resolutionSchlichting, Ilme; Berendzen, Joel; Chu, Kelvin; Stock, Ann M.; Maves, Shelley A.; Benson, David E.; Sweet, Robert M.; Ringe, Dagmar; Petsko, Gregory A.; Sligar, Stephen G.Science (Washington, D. C.) (2000), 287 (5458), 1615-1622CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Members of the cytochrome P 450 superfamily catalyze the addn. of mol. oxygen to nonactivated hydrocarbons at physiol. temp., a reaction that requires high temp. to proceed to the absence of a catalyst. Structures were obtained for three intermediates in the hydroxylation reaction of camphor by P450cam with trapping techniques and cryo-crystallog. The structure of the ferrous dioxygen adduct of P450cam was detd. with 0.91 angstrom wavelength x-rays; irradn. with 1.5 angstrom x-rays results in breakdown of the dioxygen mol. to an intermediate that would be consistent with an oxyferryl species. The structures show conformational changes in several important residues and reveal a network of bound water mols. that may provide the protons needed for the reaction.
- 63Qiu, Z.; Kuhn, B.; Aebi, J.; Lin, X.; Ding, H.; Zhou, Z.; Xu, Z.; Xu, D.; Han, L.; Liu, C.; Qiu, H.; Zhang, Y.; Haap, W.; Riemer, C.; Stahl, M.; Qin, N.; Shen, H. C.; Tang, G. Discovery of fluoromethylketone-based peptidomimetics as covalent ATG4B (Autophagin-1) inhibitors. ACS Med. Chem. Lett. 2016, 7, 802– 806, DOI: 10.1021/acsmedchemlett.6b00208[ACS Full Text], [CAS], Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVKisLrL&md5=5068eb96c6fae28342766f54010c5ebeDiscovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) InhibitorsQiu, Zongxing; Kuhn, Bernd; Aebi, Johannes; Lin, Xianfeng; Ding, Haiyuan; Zhou, Zheng; Xu, Zhiheng; Xu, Danqing; Han, Li; Liu, Cheng; Qiu, Hongxia; Zhang, Yuxia; Haap, Wolfgang; Riemer, Claus; Stahl, Martin; Qin, Ning; Shen, Hong C.; Tang, GuozhiACS Medicinal Chemistry Letters (2016), 7 (8), 802-806CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)ATG4B or autophagin-1 is a cysteine protease that cleaves ATG8 family proteins. ATG4B plays essential roles in the autophagosome formation and the autophagy pathway. Herein we disclose the design and structural modifications of a series of fluoromethylketone (FMK)-based peptidomimetics as highly potent ATG4B inhibitors. Their structure-activity relationship (SAR) and protease selectivity are also discussed.
- 64Davies, C. W.; Chaney, J.; Korbel, G.; Ringe, D.; Petsko, G. A.; Ploegh, H.; Das, C. The co-crystal structure of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) with a tripeptide fluoromethyl ketone (Z-VAE(OMe)-FMK). Bioorg. Med. Chem. Lett. 2012, 22, 3900– 3904, DOI: 10.1016/j.bmcl.2012.04.124[Crossref], [PubMed], [CAS], Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XntlSrs7k%253D&md5=1c5dd6939599cd6dc7582871d039209cThe co-crystal structure of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) with a tripeptide fluoromethyl ketone (Z-VAE(OMe)-FMK)Davies, Christopher W.; Chaney, Joseph; Korbel, Gregory; Ringe, Dagmar; Petsko, Gregory A.; Ploegh, Hidde; Das, ChittaranjanBioorganic & Medicinal Chemistry Letters (2012), 22 (12), 3900-3904CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)UCHL1 is a 223 amino acid member of the UCH family of deubiquitinating enzymes (DUBs), found abundantly and exclusively expressed in neurons and the testis in normal tissues. Two naturally occurring variants of UCHL1 are directly involved in Parkinson's disease (PD). Not only has UCHL1 been linked to PD, but it has oncogenic properties, having been found abnormally expressed in lung, pancreatic, and colorectal cancers. Although inhibitors of UCHL1 have been described previously the co-crystal structure of the enzyme bound to any inhibitor has not been reported. Herein, we report the x-ray structure of UCHL1 co-crystd. with a peptide-based fluoromethylketone inhibitor, Z-VAE(OMe)-FMK (VAEFMK) at 2.35 Å resoln. The co-crystal structure reveals that the inhibitor binds in the active-site cleft, irreversibly modifying the active-site cysteine; however, the catalytic histidine is still misaligned as seen in the native structure, suggesting that the inhibitor binds to an inactive form of the enzyme. Our structure also reveals that the inhibitor approaches the active-site cleft from the opposite side of the crossover loop as compared to the direction of approach of ubiquitin's C-terminal tail, thereby occupying the P1' (leaving group) site, a binding site perhaps used by the unknown C-terminal extension of ubiquitin in the actual in vivo substrate(s) of UCHL1. This structure provides a view of mol. contacts at the active-site cleft between the inhibitor and the enzyme as well as furnishing structural information needed to facilitate further design of inhibitors targeted to UCHL1 with high selectivity and potency.
- 65Powers, J. C.; Asgian, J. L.; Ekici, O. D.; James, K. E. Irreversible inhibitors of serine, cysteine, and threonine proteases. Chem. Rev. 2002, 102, 4639– 4750, DOI: 10.1021/cr010182v[ACS Full Text], [CAS], Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XosFSnsr4%253D&md5=eb21ff0297b87537a7d459c92f13c75dIrreversible inhibitors of serine, cysteine, and threonine proteasesPowers, James C.; Asgian, Juliana L.; Ekici, Oezlem Dogan; James, Karen EllisChemical Reviews (Washington, DC, United States) (2002), 102 (12), 4639-4750CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Topics discussed include alkylating, acylating, phosphonylating, and sulfonylating agents.
- 66Heimann, D.; Borgel, F.; de Vries, H.; Bachmann, K.; Rose, V. E.; Frehland, B.; Schepmann, D.; Heitman, L. H.; Wunsch, B. Optimization of pharmacokinetic properties by modification of a carbazole-based cannabinoid receptor subtype 2 (CB2) ligand. Eur. J. Med. Chem. 2018, 143, 1436– 1447, DOI: 10.1016/j.ejmech.2017.10.049[Crossref], [PubMed], [CAS], Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslyrt7jN&md5=4a5e0eb0666d6be4bfd948f2abe58ac9Optimization of pharmacokinetic properties by modification of a carbazole-based cannabinoid receptor subtype 2 (CB2) ligandHeimann, Dominik; Boergel, Frederik; de Vries, Henk; Bachmann, Kim; Rose, Victoria E.; Frehland, Bastian; Schepmann, Dirk; Heitman, Laura H.; Wuensch, BernhardEuropean Journal of Medicinal Chemistry (2018), 143 (), 1436-1447CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Recently, the development of the fluorinated PET tracer [18F]1a for imaging of CB2 receptors in the central nervous system was reported. [18F]1a showed high CB2 affinity and selectivity over the CB1 subtype, but rapid biotransformation in mice. In addn. to the amide hydrolysis, oxidative N-dealkylation and carbazole oxidn. were postulated as main metabolic pathways. Based on these results, novel carbazole derivs. with addnl. 6-substituents (23a, 24a), modified hydrogenation state (26a) and enlarged fluoroalkyl substituent (13a, 13b) were synthesized and pharmacol. evaluated. The key step in the synthesis of substituted carbazoles 23a, 24a and 26a was a Fischer indole synthesis. Nucleophilic substitution of tosylated lactate 5 by carbazole anion provided the fluoroisopropyl derivs. 13a and 13b. Partial hydrogenation of the arom. carbazole system (26a) was not tolerated by the CB2 receptor. A methylsulfonyl moiety in 6-position (24a) led to considerably reduced CB2 affinity, whereas a 6-methoxy moiety (23a) was well tolerated. An addnl. Me moiety in the fluoroethyl side chain of 1a resulted in fluoroisopropyl derivs. 13 with unchanged high CB2 affinity and CB2: CB1 selectivity. Compared with the fluoroethyl deriv. 1a, the carbazole N-atom of the fluoroisopropyl deriv. 13a (Ki(CB2) = 2.9 nM) is better shielded against the attack of CYP enzymes as formation of N-oxides was not obsd. and N-dealkylation took place to a less amt.
- 67Frolova, A. D.; Kuznetsova, E. E. [Transformations of fluorinated ketones in the organism]. Gig. Tr. Prof. Zabol. 1971, 15, 57– 59[PubMed], [CAS], Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3MXks1WmtLY%253D&md5=bc8bc7a38faed36cde566f7d633cb291Transformation of fluorinated ketones in the organismFrolova, A. D.; Kuznetsova, E. E.Gigiena Truda i Professional'nye Zabolevaniya (1971), 15 (6), 57-9CODEN: GTPZAB; ISSN:0016-9919.Acute intoxication by monofluoroacetone in rats caused accumulation of citric acid in the kidneys (fluoroacetate type of action). A toxic concn. of perfluoroacetone dihydrate increased in 6 hr the excretion of glucuronides by urine. The synthesis of fluorinated keto acid or fluorinated secondary alc. is presumed.
- 68Mazurek, U.; Koszinowski, K.; Schwarz, H. C–F bond activation in fluorinated carbonyl compounds by chromium monocations in the gas phase. Organometallics 2003, 22, 218– 225, DOI: 10.1021/om020646v[ACS Full Text], [CAS], Google Scholar68https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xps1Wqtbs%253D&md5=4bb2a82960e596a8697d2a59cdba6c1bC-F Bond Activation in Fluorinated Carbonyl Compounds by Chromium Monocations in the Gas PhaseMazurek, Ulf; Koszinowski, Konrad; Schwarz, HelmutOrganometallics (2003), 22 (2), 218-225CODEN: ORGND7; ISSN:0276-7333. (American Chemical Society)The Cr+-assisted hydrolytic C-F bond activation in the gas phase reported recently for hexafluoroacetone applies also to other fluorinated carbonyl compds. C-F bond hydrolysis is obsd. for monofluoroacetone, 1,1,1-trifluoroacetone, pentafluorobenzaldehyde, and 2,3,4,5,6-pentafluoroacetophenone. However, the diversity of the carbonyl group's substituents is paralleled by an increase of the hitherto small no. of reaction channels, thus allowing for alternative ways of C-F bond activation as well. Both complexation and C-F bond activation of the org. substrates in the gas phase have been investigated by FT-ICR mass spectrometry.
- 69Arellano, M.; Malet-Martino, M.; Martino, R.; Gires, P. The anti-cancer drug 5-fluorouracil is metabolized by the isolated perfused rat liver and in rats into highly toxic fluoroacetate. Br. J. Cancer 1998, 77, 79– 86, DOI: 10.1038/bjc.1998.12[Crossref], [PubMed], [CAS], Google Scholar69https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXoslertg%253D%253D&md5=64430ef44e3f0016a1617e093fa36c14The anti-cancer drug 5-fluorouracil is metabolized by the isolated perfused rat liver and in rats into highly toxic fluoroacetateArellano, M.; Malet-Martino, M.; Martino, R.; Gires, P.British Journal of Cancer (1998), 77 (1), 79-86CODEN: BJCAAI; ISSN:0007-0920. (Churchill Livingstone)The authors report the first demonstration of the biotransformation of the anti-cancer drug 5-fluorouracil (FU) into two new metabolites, α-fluoro-β-hydroxypropionic acid (FHPA) and fluoroacetate (FAC), in the isolated perfused rat liver (IPRL) and in the rat in vivo. IPRL was perfused with solns. of pure FU at two doses, 15 or 45 mg kg-1 body wt., and rats were injected i.p. with 180 mg of FU kg-1 body wt. Fluorine-19 NMR anal. of perfusates from IPRL and rat urine showed the presence of the normal metabolites of FU and low amts. of FHPA (0.4% or 0.1% of injected FU in perfusates from IPRL treated with 15 or 45 mg of FU kg-1 body wt., resp.; 0.08% of the injected FU in rat urine) and FAC (0.1% or 0.03% of injected FU in perfusates from IPRL treated with 15 or 45 mg of FU kg-1 body wt., resp.; 0.003% of the injected FU in rat urine). IPRL was also perfused with a soln. of α-fluoro-β-alanine (FBAL) hydrochloride at 16.6 mg kg-1 body wt. dose equiv. to 15 mg of FU kg-1 body wt. Low amts. of FHPA (0.2% of injected FBAL) and FAC (0.07%) were detected in perfusates, thus demonstrating that FHPA and FAC arise from FBAL catabolism. As FAC is a well-known cardiotoxic poison, and FHPA is also cardiotoxic at high doses, the cardiotoxicity of FU might stem from at least two sources. The first one, established in previous papers, is the presence in com. solns. of FU of degrdn. products of FU that are metabolized into FHPA and FAC; these are formed over time in the basic medium necessary to dissolve the drug. The second, demonstrated in the present study, is the metab. of FU itself into the same compds.
- 70Malet-Martino, M.; Gilard, V.; Desmoulin, F.; Martino, R. Fluorine nuclear magnetic resonance spectroscopy of human biofluids in the field of metabolic studies of anticancer and antifungal fluoropyrimidine drugs. Clin. Chim. Acta 2006, 366, 61– 73, DOI: 10.1016/j.cca.2005.10.013[Crossref], [PubMed], [CAS], Google Scholar70https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XitVKhtb0%253D&md5=d064f058075c536e08c00faf85dd4ac2Fluorine nuclear magnetic resonance spectroscopy of human biofluids in the field of metabolic studies of anticancer and antifungal fluoropyrimidine drugsMalet-Martino, Myriam; Gilard, Veronique; Desmoulin, Franck; Martino, RobertClinica Chimica Acta (2006), 366 (1-2), 61-73CODEN: CCATAR; ISSN:0009-8981. (Elsevier Ltd.)A review with refs. Fluorine-19 NMR (19F NMR) spectroscopy provides a highly specific tool for the detection, identification and quantification of fluorine-contg. drugs and their metabolites in biofluids. The value and difficulties encountered in investigations on drug metab. are first discussed. Then the metab. of three fluoropyrimidines in clin. use, 5-fluorouracil, 5-fluorocytosine and capecitabine are reported. Besides the parent drug and the already known fluorinated metabolites, 12 new metabolites were identified for the first time with 19F NMR in human biofluids. Nine of them can only be obsd. with this technique: fluoride ion, N-carboxy-α-fluoro-β-alanine, α-fluoro-β-alanine conjugate with deoxycholic acid, 2-fluoro-3-hydroxypropanoic acid, fluoroacetic acid, O2-β-glucuronide of fluorocytosine, fluoroacetaldehyde hydrate and its adduct with urea, fluoromalonic acid semi-aldehyde adducts with urea. This emphasizes the high anal. potential of 19F NMR for the furtherance in the understanding of fluoropyrimidine catabolic pathways. 19F NMR should also play a role in the therapeutic monitoring of FU and its prodrugs in specific groups of patients, e.g. hemodialyzed patients or patients with deficiency in FU catabolic enzymes.
- 71Derissen, E. J.; Jacobs, B. A.; Huitema, A. D.; Rosing, H.; Schellens, J. H.; Beijnen, J. H. Exploring the intracellular pharmacokinetics of the 5-fluorouracil nucleotides during capecitabine treatment. Br. J. Clin. Pharmacol. 2016, 81, 949– 957, DOI: 10.1111/bcp.12877[Crossref], [PubMed], [CAS], Google Scholar71Exploring the intracellular pharmacokinetics of the 5-fluorouracil nucleotides during capecitabine treatmentDerissen, Ellen J. B.; Jacobs, Bart A. W.; Huitema, Alwin D. R.; Rosing, Hilde; Schellens, Jan H. M.; Beijnen, Jos H.British Journal of Clinical Pharmacology (2016), 81 (5), 949-957CODEN: BCPHBM; ISSN:1365-2125. (Wiley-Blackwell)Aim : Three intracellularly formed metabolites are responsible for the antineoplastic effect of capecitabine: 5-fluorouridine 5'-triphosphate (FUTP), 5-fluoro-2'-deoxyuridine 5'-triphosphate (FdUTP), and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). The objective of this study was to explore the pharmacokinetics of these intracellular metabolites during capecitabine treatment. Methods : Serial plasma and peripheral blood mononuclear cell (PBMC) samples were collected from 13 patients treated with capecitabine 1000 mg QD (group A) and eight patients receiving capecitabine 850 mg m-2 BID for fourteen days, every three weeks (group B). Samples were collected on day 1 and, for four patients of group B, also on day 14. The capecitabine and 5-fluorouracil (5-FU) plasma concns. and intracellular metabolite concns. were detd. using LC-MS/MS. Pharmacokinetic parameters were estd. using non-compartmental anal. Results : Only FUTP could be measured in the PBMC samples. The FdUTP and FdUMP concns. were below the detection limits (LOD). No significant correlation was found between the plasma 5-FU and intracellular FUTP exposure. The FUTP concn.-time profiles demonstrated considerable inter-individual variation and accumulation of the metabolite in PBMCs. FUTP levels ranged betweenhttps://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmtF2mu7s%253D&md5=d31ab3b2043d45bbe86305e934247ab072Desmoulin, F.; Gilard, V.; Malet-Martino, M.; Martino, R. Metabolism of capecitabine, an oral fluorouracil prodrug: (19)F NMR studies in animal models and human urine. Drug Metab. Dispos. 2002, 30, 1221– 1229, DOI: 10.1124/dmd.30.11.1221[Crossref], [PubMed], [CAS], Google Scholar72https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XotFyku74%253D&md5=ca4db01fd109f974459848cd82a6e974Metabolism of capecitabine, an oral fluorouracil prodrug: 19F NMR studies in animal models and human urineDesmoulin, Franck; Gilard, Veronique; Malet-Martino, Myriam; Martino, RobertDrug Metabolism and Disposition (2002), 30 (11), 1221-1229CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)Capecitabine (Xeloda; CAP) is a recently developed oral antineoplastic prodrug of 5-fluorouracil (5-FU) with enhanced tumor selectivity. Previous studies have shown that CAP activation follows a pathway with three enzymic steps and two intermediary metabolites, 5'-deoxy-5-fluorocytidine (5'-DFCR) and 5'-deoxy-5-fluorouridine (5'-DFUR), to form 5-FU preferentially in tumor tissues. In the present work, we investigated all fluorinated compds. present in liver, bile, and perfusate medium of isolated perfused rat liver (IPRL) and in liver, plasma, kidneys, bile, and urine of healthy rats. Moreover, data obtained from rat urine were compared with those from mice and human urine. According to a low cytidine deaminase (3.5.4.5) activity in rats, 5'-DFCR was by far the main product in perfusate medium from IPRL and plasma and urine from rats. Liver and circulating 5'-DFCR in perfusate and plasma equilibrated at the same concn. value in the range 25 to 400 μM, which supports the involvement of es-type nucleoside transporter in the liver. 5'-DFUR and α-fluoro-β-ureidopropionic acid (FUPA) + α-fluoro-β-alanine (FBAL) were the main products in urine of mice, making up 23 to 30% of the administered dose vs. 3 to 4% in rat. In human urine, FUPA + FBAL represented 50% of the administered dose, 5'-DFCR 10%, and 5'-DFUR 7%. Since fluorine-19 NMR spectroscopy gives an overview of all the fluorinated compds. present in a sample, we obsd. the following unreported metabolites of CAP:. 1) 5-Fluorocytosine and its hydroxylated metabolite, 5-fluoro-6-hydroxycytosine,. 2) Fluoride ion,. 3) 2-Fluoro-3-hydroxypropionic acid and fluoroacetate, and. 4) A glucuroconjugate of 5'-DFCR.73Carreras, C. W.; Santi, D. V. The catalytic mechanism and structure of thymidylate synthase. Annu. Rev. Biochem. 1995, 64, 721– 762, DOI: 10.1146/annurev.bi.64.070195.003445[Crossref], [PubMed], [CAS], Google Scholar73https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXmsl2qtrg%253D&md5=3fa2b583e048196a12193b27f9533770The catalytic mechanism and structure of thymidylate synthaseCarreras, Christopher W.; Santi, Daniel V.Annual Review of Biochemistry (1995), 64 (), 721-62CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)A review, with 193 refs. Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the reductive methylation of dUMP by CH2H4folate to produce dTMP and H2folate. Knowledge of the catalytic mechanism and structure of TS has increased substantially over recent years. Major advances were derived from crystal structures of TS bound to various ligands, the ability to overexpress TS in heterologous hosts, and the numerous mutants that have been prepd. and analyzed. These advances, coupled with previous knowledge, have culminated in an in-depth understanding of many important mol. details of the reaction. The authors review aspects of TS catalysis that are most pertinent to understanding the current status of the structure and catalytic mechanism of the enzyme. Included is a discussion of available sources and assays for TS, a description of the enzyme's chem. mechanism and crystal structure, and a summary of data obtained from mutagenesis expts.74Zhang, X.; Zhang, N.; Chen, G.; Turpoff, A.; Ren, H.; Takasugi, J.; Morrill, C.; Zhu, J.; Li, C.; Lennox, W.; Paget, S.; Liu, Y.; Almstead, N.; George Njoroge, F.; Gu, Z.; Komatsu, T.; Clausen, V.; Espiritu, C.; Graci, J.; Colacino, J.; Lahser, F.; Risher, N.; Weetall, M.; Nomeir, A.; Karp, G. M. Discovery of novel HCV inhibitors: synthesis and biological activity of 6-(indol-2-yl)pyridine-3-sulfonamides targeting hepatitis C virus NS4B. Bioorg. Med. Chem. Lett. 2013, 23, 3947– 3953, DOI: 10.1016/j.bmcl.2013.04.049[Crossref], [PubMed], [CAS], Google Scholar74https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXnvFSisrw%253D&md5=c4ea419ba26dea48358a3036caf48abfDiscovery of novel HCV inhibitors: Synthesis and biological activity of 6-(indol-2-yl)pyridine-3-sulfonamides targeting hepatitis C virus NS4BZhang, Xiaoyan; Zhang, Nanjing; Chen, Guangming; Turpoff, Anthony; Ren, Hongyu; Takasugi, James; Morrill, Christie; Zhu, Jin; Li, Chunshi; Lennox, William; Paget, Steven; Liu, Yalei; Almstead, Neil; George Njoroge, F.; Gu, Zhengxian; Komatsu, Takashi; Clausen, Valerie; Espiritu, Christine; Graci, Jason; Colacino, Joseph; Lahser, Fred; Risher, Nicole; Weetall, Marla; Nomeir, Amin; Karp, Gary M.Bioorganic & Medicinal Chemistry Letters (2013), 23 (13), 3947-3953CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)A novel series of 6-(indol-2-yl)pyridine-3-sulfonamides I [R1 = CHF2O, c-Pr; R2 = i-Pr, H, CH(CH2F)2, etc.] was prepd. and evaluated for their ability to inhibit HCV RNA replication in the HCV replicon cell culture assay. Preliminary optimization of this series furnished compds. with low nanomolar potency against the HCV genotype 1b replicon. Among these, compd. I [R1 = CHF2O; R2 = CH(CH2F)2] was identified as a potent HCV replicon inhibitor (EC50 = 4 nM) with a selectivity index with respect to cellular GAPDH of more than 2500. Further, compd. I [R1 = CHF2O; R2 = CH(CH2F)2] had a good pharmacokinetic profile in rats with an IV half-life of 6 h and oral bioavailability (F) of 62%. Selection of HCV replicon resistance identified an amino acid substitution in HCV NS4B that confers resistance to these compds. These compds. hold promise as a new chemotype with anti-HCV activity mediated through an underexploited viral target.75Zhang, N.; Zhang, X.; Zhu, J.; Turpoff, A.; Chen, G.; Morrill, C.; Huang, S.; Lennox, W.; Kakarla, R.; Liu, R.; Li, C.; Ren, H.; Almstead, N.; Venkatraman, S.; Njoroge, F. G.; Gu, Z.; Clausen, V.; Graci, J.; Jung, S. P.; Zheng, Y.; Colacino, J. M.; Lahser, F.; Sheedy, J.; Mollin, A.; Weetall, M.; Nomeir, A.; Karp, G. M. Structure-activity relationship (SAR) optimization of 6-(indol-2-yl)pyridine-3-sulfonamides: identification of potent, selective, and orally bioavailable small molecules targeting hepatitis C (HCV) NS4B. J. Med. Chem. 2014, 57, 2121– 2135, DOI: 10.1021/jm401621g[ACS Full Text], [CAS], Google Scholar75https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVajtbjL&md5=135ebcfa314f96d786711155128b78c4Structure-Activity Relationship (SAR) Optimization of 6-(Indol-2-yl)pyridine-3-sulfonamides: Identification of Potent, Selective, and Orally Bioavailable Small Molecules Targeting Hepatitis C (HCV) NS4BZhang, Nanjing; Zhang, Xiaoyan; Zhu, Jin; Turpoff, Anthony; Chen, Guangming; Morrill, Christie; Huang, Song; Lennox, William; Kakarla, Ramesh; Liu, Ronggang; Li, Chunshi; Ren, Hongyu; Almstead, Neil; Venkatraman, Srikanth; Njoroge, F. George; Gu, Zhengxian; Clausen, Valerie; Graci, Jason; Jung, Stephen P.; Zheng, Yingcong; Colacino, Joseph M.; Lahser, Fred; Sheedy, Josephine; Mollin, Anna; Weetall, Marla; Nomeir, Amin; Karp, Gary M.Journal of Medicinal Chemistry (2014), 57 (5), 2121-2135CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Nonracemic (aminosulfonyl)pyridinyl indolecarbonitriles such as I were prepd. as inhibitors of the hepatitis C viral protein NS4B for use as antihepatitis C agents. The substitution patterns on the indole rings were modified to limit oxidative metab. of the indolecarbonitriles and to avoid potential liver damage and cytochrome P450 inhibition; the compds. were also optimized to improve their oral bioavailabilities. I was potent against the HCV 1b replicon, with an EC50 value of 2 nM and a selectivity of >5000 with respect to cellular glyceraldehyde-3-phosphate dehydrogenase. I had a favorable pharmacokinetic profile with oral bioavailabilities of 62%, 78%, and 18% in rats, dogs, and monkeys, resp., and favorable tissue distribution properties (liver to plasma exposure ratio in rats of 25).76Appelman, L. M.; Woutersen, R. A.; Feron, V. J. Inhalation toxicity of acetaldehyde in rats. I. Acute and subacute studies. Toxicology 1982, 23, 293– 307, DOI: 10.1016/0300-483X(82)90068-3[Crossref], [PubMed], [CAS], Google Scholar76https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XkvVKmu74%253D&md5=8af732af35478d6f36313c8342c7bc15Inhalation toxicity of acetaldehyde in rats. I. Acute and subacute studiesAppelman, L. M.; Woutersen, R. A.; Feron, V. J.Toxicology (1982), 23 (4), 293-307CODEN: TXCYAC; ISSN:0300-483X.The 4-h median lethal concn. of AcH [75-07-0] in rats was 13,300 ppm (24.0 g/m3 air). In a 4-wk study, groups of male and female rats were exposed to 0, 400, 1000, 2200 or 5000 ppm AcH for 6 h/day, 5 days/wk. Treatment-related changes obsd. at the 5000 ppm level included dyspnea and excitation during the 1st 30 min of each exposure, yellow-brown fur, severe growth retardation, more neutrophils and less lymphocytes in the blood, a reduced prodn. of urine with a high d., increased lung wts., and severe degenerative, hyperplastic and metaplastic changes of the nasal, laryngeal and tracheal epithelium. Major lesions seen at 1000 and 2200 ppm comprised growth retardation and an increased prodn. of urine in males, slight to moderate degeneration with or without hyper- and metaplasia of the nasal epithelium, and only at 220 ppm, minimal epithelial changes in the larynx and trachea. The only change obsd. at the 400 ppm level that could be attributed to AcH was slight degeneration of the nasal olfactory epithelium seen as loss of microvilli and thinning and disarrangement of the layer of epithelial cells.77Woutersen, R. A.; Appelman, L. M.; Van der Heijden, C. A. Inhalation toxicity of acetaldehyde in rats. II. Carcinogenicity study: interim results after 15 months. Toxicology 1984, 31, 123– 133, DOI: 10.1016/0300-483X(84)90004-0[Crossref], [PubMed], [CAS], Google Scholar77https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXltVGmsbY%253D&md5=8a65ee868ccab29ae05d811a3898a1a1Inhalation toxicity of acetaldehyde in rats. II. Carcinogenicity study: interim results after 15 monthsWoutersen, R. A.; Appelman, L. M.; Feron, V. J.; Van der Heijden, C. A.Toxicology (1984), 31 (2), 123-33CODEN: TXCYAC; ISSN:0300-483X.Male and female rats were exposed to AcH [75-07-0] vapor at 0, 750, 1500 and 3000/1000 ppm during 6 h/day, 5 days/wk for up to 27 mo. During the 1st 15 mo of the study, major compd.-related lesions occurred in the nose and larynx. The nasal lesions comprised: (1) degenerative changes of the olfactory epithelium at all dose levels, frequently accompanied by focal hyperplasia of basal cells and thickening of the submucosa with loss of Bowman's glands and nerve bundles in the dorsomedial region, (2) stratified squamous metaplasia of the respiratory epithelium lining the caudoventral part of the nasal septum and the inner aspect of the ventral endoturbinates often accompanied by severe keratinization and occasionally by papillomatous hyperplasia, almost exclusively obsd. at the top-dose level, and (3) malignant tumors (squamous cell carcinomas and adenocarcinomas) at all dose levels. Hyperplasia and keratinized stratified squamous metaplasia of the laryngeal epithelium were seen at the 2 highest dose levels.78Woutersen, R. A.; Appelman, L. M.; Van Garderen-Hoetmer, A.; Feron, V. J. Inhalation toxicity of acetaldehyde in rats. III. Carcinogenicity study. Toxicology 1986, 41, 213– 231, DOI: 10.1016/0300-483X(86)90201-5[Crossref], [PubMed], [CAS], Google Scholar78https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28Xmt1Skt70%253D&md5=6880976e51089259ca4dfc12bd17ea9bInhalation toxicity of acetaldehyde in rats. III. Carcinogenicity studyWoutersen, R. A.; Appelman, L. M.; Van Garderen-Hoetmer, A.; Feron, V. J.Toxicology (1986), 41 (2), 213-31CODEN: TXCYAC; ISSN:0300-483X.Male and female rats were exposed to AcH [75-07-0] vapor at nominal concns. of 0, 750, 1500, or 3000 ppm (gradually reduced to 1000 ppm during the 1st 52 wk) during 6 h/day, 5 days/wk for ≤28 mo. Major compd.-related effects included increased mortality, growth retardation, nasal tumors, and nonneoplastic nasal changes in each of the test groups. The treatment-related nasal changes comprised: (1) degeneration, hyperplasia, metaplasia, and adenocarcinomas of the olfactory epithelium at all exposure levels; (2) squamous metaplasia accompanied by slight to severe keratinization and squamous cell carcinomas of the respiratory epithelium at the 2 highest exposure levels; and (3) slight to severe rhinitis and sinusitis in top-concn. rats. In the larynx hyperplasia and keratinized squamous metaplasia of the epithelium in the vocal cord region were seen in many rats of the mid- and top-concn. groups. One female rat of the 1500-ppm group had developed a laryngeal carcinoma in situ. Evidently, AcH is both cytotoxic and carcinogenic to the nasal mucosa of rats.79Setshedi, M.; Wands, J. R.; de la Monte, S. M. Acetaldehyde adducts in alcoholic liver disease. Oxid. Med. Cell. Longevity 2010, 3, 178– 185, DOI: 10.4161/oxim.3.3.12288[Crossref], [PubMed], [CAS], Google Scholar79https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3cjlsF2ntg%253D%253D&md5=52da45f51f23fc86bfa7f854fdb70263Acetaldehyde adducts in alcoholic liver diseaseSetshedi Mashiko; Wands Jack R; Monte Suzanne M de laOxidative medicine and cellular longevity (2010), 3 (3), 178-85 ISSN:.Chronic alcohol abuse causes liver disease that progresses from simple steatosis through stages of steatohepatitis, fibrosis, cirrhosis, and eventually hepatic failure. In addition, chronic alcoholic liver disease (ALD), with or without cirrhosis, increases risk for hepatocellular carcinoma (HCC). Acetaldehyde, a major toxic metabolite, is one of the principal culprits mediating fibrogenic and mutagenic effects of alcohol in the liver. Mechanistically, acetaldehyde promotes adduct formation, leading to functional impairments of key proteins, including enzymes, as well as DNA damage, which promotes mutagenesis. Why certain individuals who heavily abuse alcohol, develop HCC (7.2-15%) versus cirrhosis (15-20%) is not known, but genetics and co-existing viral infection are considered pathogenic factors. Moreover, adverse effects of acetaldehyde on the cardiovascular system and hematologic systems leading to ischemia, heart failure, and coagulation disorders, can exacerbate hepatic injury and increase risk for liver failure. Herein, we review the role of acetaldehyde adducts in the pathogenesis of chronic ALD and HCC.80Nagasawa, H. T.; Valentekovich, R. J.; Nagasawa, S. G.; Nagasawa, R. H. Sequestration and elimination of toxic aldehydes. Chem. Res. Toxicol. 2020, DOI: 10.1021/acs.chemrestox.9b0037381Ernstgard, L.; Iregren, A.; Sjogren, B.; Johanson, G. Acute effects of exposure to vapours of acetic acid in humans. Toxicol. Lett. 2006, 165, 22– 30, DOI: 10.1016/j.toxlet.2006.01.010[Crossref], [PubMed], [CAS], Google Scholar81https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XlvF2gu74%253D&md5=04ea02fe480a664b494cc0ff8525619aAcute effects of exposure to vapours of acetic acid in humansErnstgard, Lena; Iregren, Anders; Sjoegren, Bengt; Johanson, GunnarToxicology Letters (2006), 165 (1), 22-30CODEN: TOLED5; ISSN:0378-4274. (Elsevier B.V.)Acetic acid is used in plastics, chem. and pharmaceutical industries. Despite a widespread use, information of possible health effects is sparse. The aim of this study was to evaluate acute irritation during controlled exposure to vapors of acetic acid. Six female and 6 male healthy volunteers were exposed to 0 ppm (control exposure), 5 and 10 ppm acetic acid vapor for 2 h at rest in a balanced order. Subjective ratings of nasal irritation and smell increased significantly with exposure level. Except for smell, all av. ratings at 10 ppm were at the lower end of the 0-100 mm visual analog scale, and did not exceed the verbal expression "somewhat" (26 mm). No effects on pulmonary function, nasal swelling, nasal airway resistance or plasma inflammatory markers (C-reactive protein, and interleukin-6), measured before and after exposure, were seen. There was a non-significant tendency to increased blinking frequency, as measured continuously during exposure, after exposure to 10 ppm acetic acid. In conclusion, our study suggests a mild irritative effect at 10 ppm acetic acid.82Zeiger, E.; Anderson, B.; Haworth, S.; Lawlor, T.; Mortelmans, K. Salmonella mutagenicity tests: V. Results from the testing of 311 chemicals. Environ. Mol. Mutagen. 1992, 19 (S21), 2– 141, DOI: 10.1002/em.2850190603[Crossref], [PubMed], [CAS], Google Scholar82https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xks1Cmurw%253D&md5=ef3f19f34f891ad579448473326a7182Salmonella mutagenicity tests: V. Results from the testing of 311 chemicalsZeiger, Errol; Anderson, Beth; Haworth, Steve; Lawlor, Timothy; Mortelmans, KristienEnvironmental and Molecular Mutagenesis (1992), 19 (Suppl. 21), 2-141CODEN: EMMUEG; ISSN:0893-6692.Three hundred eleven chems. were tested under code, for mutagenicity, in S. typhimurium; 35 of the chems. were tested more than once in the same or different labs. The tests were conducted using a preincubation protocol in the absence of exogenous metabolic activation, and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters. Some of the volatile chems. were also tested in desiccators. A total of 120 chems. were mutagenic or weakly mutagenic, 3 were judged questionable, and 172 were nonmutagenic. The remaining 16 chems. produced different responses in the two or three labs. in which they were tested. The results and data from these tests are presented.83Morita, T.; Takeda, K.; Okumura, K. Evaluation of clastogenicity of formic acid, acetic acid and lactic acid on cultured mammalian cells. Mutat. Res., Genet. Toxicol. Test. 1990, 240, 195– 202, DOI: 10.1016/0165-1218(90)90058-A[Crossref], [PubMed], [CAS], Google Scholar83https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXhsFemsrs%253D&md5=5c1f988071dede0a3d0f84b6e3272126Evaluation of clastogenicity of formic acid, acetic acid, and lactic acid on cultured mammalian cellsMorita, Takeshi; Takeda, Kenzo; Okumura, KazuoMutation Research, Genetic Toxicology Testing (1990), 240 (3), 195-202CODEN: MRGTE4; ISSN:0165-1218.Using Chinese hamster ovary K1 cells, chromosomal aberration tests were carried out with formic acid, acetic acid and lactic acid, and the relation between the pH of the medium and the clastogenic activity was examd. The medium used was Ham's F12 supplemented with 17 mM NaHCO3 and 10% fetal calf serum. All the acids induced chromosomal aberrations at the initial pH of ca. 6.0 or below (∼10-14 mM of each acid) both with and without S9 mix. Exposure of cells to about pH 5.7 or below (∼12-16 mM of each acid) was toxic. When the culture medium was first acidified with each of the acids and then neutralized to pH 6.4 or pH 7.2 with NaOH, no clastogenic activity was obsd. Using F12 medium supplemented with 34 mM NaHCO3 as a buffer, no clastogenic activity was obsd. at doses up to 25 mM of the acids (initial pH 5.8-6.0). However, ∼10% of the cells had aberrations at pH 5.7 or below (27.5-32.5 mM of each acid). Furthermore, when 30 mM HEPES was used as a buffer, chromosomal aberrations were not induced at doses up to 20 mM formic acid and acetic acid (initial pH 7.0-7.1), and at doses up to 30 mM lactic acid (initial pH 6.6). In the initial pH range of 6.4-6.7 (25-32.5 mM of each acid), chromosomal aberrations were obsd. The above results show that the acids themselves are nonclastogenic, and the pseudopos. reactions attributable to nonphysiol. pH could be eliminated by either neutralization of the medium or enhancement of its buffering ability.84Rotstein, J. B.; Slaga, T. J. Acetic acid, a potent agent of tumor progression in the multistage mouse skin model for chemical carcinogenesis. Cancer Lett. 1988, 42, 87– 90, DOI: 10.1016/0304-3835(88)90243-1[Crossref], [PubMed], [CAS], Google Scholar84https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXjsFWgtQ%253D%253D&md5=833c940fb86167f0d7e62f696656364aAcetic acid, a potent agent of tumor progression in the multistage mouse skin model for chemical carcinogenesisRotstein, J. B.; Slaga, T. J.Cancer Letters (Shannon, Ireland) (1988), 42 (1-2), 87-90CODEN: CALEDQ; ISSN:0304-3835.Acetic acid, a very weak tumor promoter in the multistage mouse skin model for chem. carcinogenesis, was found to be very effective at enhancing cancer development, when applied during the progression phase of the model. Papilloma-bearing mice when repeatedly treated with acetic acid had a greater carcinoma incidence and a greater conversion of papillomas to carcinomas than vehicle-treated mice. Selective cytotoxicity is discussed as a possible mechanism.85Hunter, E. S., 3rd; Rogers, E. H.; Schmid, J. E.; Richard, A. Comparative effects of haloacetic acids in whole embryo culture. Teratology 1996, 54, 57– 64, DOI: 10.1002/(SICI)1096-9926(199606)54:2<57::AID-TERA1>3.0.CO;2-1[Crossref], [PubMed], [CAS], Google Scholar85https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xntleju7w%253D&md5=8ee90401496b0b2652177e2858449941Comparative effects of haloacetic acids in whole embryo cultureHunter, E, Sidney, III; Rogers, E. H.; Schmid, J. E.; Richard, A.Teratology (1996), 54 (2), 57-64CODEN: TJADAB; ISSN:0040-3709. (Wiley-Liss)Three-6 somite staged CD-1 mouse embryos were exposed to acetic acid (AA) or mono- (M), di- (D), and tri- (T) substituted fluoro-(F), chloro- (C), or bromo- (B) acetic acids in whole embryo culture to evaluate the effects of these agents on development. A 24 h exposure to the haloacetic acids produced dysmorphogenesis. Effects on neural tube development ranged from prosencephalic hypoplasia to non-closure defects throughout the cranial region. Exposure to the haloacetic acid affected optic development, produced malpositioned and/or hypoplastic pharyngeal arches, and resulted in perturbation of heart development. To det. the relative toxicities of these agents, benchmark concns. were calcd. as the lower 95% confidence interval of the concn. that produced a 5% increase in neural tube defects. The benchmark concns. occurred over a wide range with DFA (5912.6 μM) and MBA (2.7 μM) at the extremes. Using the benchmark concns. to compare the chems. gives a ranking of the agents in order of increasing potency as: DFA < TFA < DCA < AA < TBA ≤ TCA < DBA < MCA < MBA. TCA and DCA have demonstrated ability to disrupt development in vivo but were among the least potent haloacetic acids in vitro. Because of the potential for widespread exposure to haloacetic acids in drinking water and the incomplete toxicity profile of these chems., further work on their developmental effects is warranted.86Richard, A. M.; Hunter, E. S., 3rd. Quantitative structure-activity relationships for the developmental toxicity of haloacetic acids in mammalian whole embryo culture. Teratology 1996, 53, 352– 360, DOI: 10.1002/(SICI)1096-9926(199606)53:6<352::AID-TERA6>3.0.CO;2-1[Crossref], [PubMed], [CAS], Google Scholar86https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xms1Gqtrk%253D&md5=29441116eaf30d4985e8a5c7084021e6Quantitative structure-activity relationships for the developmental toxicity of haloacetic acids in mammalian whole embryo cultureRichard, Ann M.; Hunter, E. Sidney, IIITeratology (1996), 53 (6), 352-360CODEN: TJADAB; ISSN:0040-3709. (Wiley-Liss)Developmental toxicity in mouse whole embryo culture assay has been reported for acetic acid (AA) and a series of ten haloacetic acids, including mono-, di-, tri-fluoro (MFA, DFA, TFA), chloro (MCA, DCA, TCA), bromo (MBA, DBA, TBA), and monoiodo (MIA) acetic acids. Benchmark concns. (BCm), calcd. as the lower 95% confidence limit of molar acid concn. producing a 5% increase in embryos with neural tube defects, provided potency ests. for development of quant. structure-activity relationships (QSARs). The best overall regression was obtained for the ten halo-acids (excluding AA) and related log(1/BCm) to the energy of the LUMO (Elumo) and acid dissocn. const. (pKa) with a correlation coeff. of r = 0.97, and a sample size-adjusted r2 = 0.92. This QSAR suggested a common basis for the mechanism of HA activity, which would imply additivity for mixts. of these acids. Examn. of QSARs for subsets of the total data set (e.g., monohaloacids) highlighted parameter relationships embedded in the total QSAR, helping to unravel the sep. contributions of Elumo and pKa to the overall potency. The relevance of these parameters is discussed in terms of postulated mechanisms of developmental toxicity involving changes in intercellular pH and redox metab. The whole embryo assay results pertain to direct embryo exposure and toxicity without the confounding influence of maternal factors. The resulting QSAR model offers possible insight into the mechanism of embryo toxicity that will hopefully contribute to understanding of the more complex, in vivo teratogenicity problem.87Lloyd, S. C.; Blackburn, D. M.; Foster, P. M. Trifluoroethanol and its oxidative metabolites: comparison of in vivo and in vitro effects in rat testis. Toxicol. Appl. Pharmacol. 1988, 92, 390– 401, DOI: 10.1016/0041-008X(88)90179-2[Crossref], [PubMed], [CAS], Google Scholar87https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXhs1aqs7k%253D&md5=a4b5f844782b0d26d54c7b75f2a88ddaTrifluoroethanol and its oxidative metabolites: comparison of in vivo and in vitro effects in rat testisLloyd, S. C.; Blackburn, D. M.; Foster, P. M. D.Toxicology and Applied Pharmacology (1988), 92 (3), 390-401CODEN: TXAPA9; ISSN:0041-008X.Trifluoroethanol (TFE) and trifluoroacetaldehyde (TFALD) decreased testis wt. 3 days after a single oral dose of 10 mg/kg. In contrast, the administration of trifluoroacetic acid (TFAA) caused no observable testicular effects. Redn. in testis wt. was accompanied by morphol. changes, involving specific damage to pachytene and dividing spermatocytes, and round spermatids. In an in vitro Sertoli/germ cell co-culture system, only TFALD had dose-related effects at concns. of 10-3 and 10-4M. There was increased germ cell loss from the cultures, particularly loss of pachytene and dividing spermatocytes, accompanied by leakage of the pachytene spermatocyte marker enzyme, lactate dehydrogenase-X. TFE and TFAA did not produce these effects in the culture system at concns. equimolar with TFALD. Thus, TFALD may play a crit. role in the development of the testis lesion seen with TFE in vivo. The effects seen both in vivo and in vitro were remarkably similar to those previously reported for another substituted alc. and its metabolites, ethylene glycol monomethyl ether. The 2 series of compds. may have a similar mode of action on rat testis.88Kaminsky, L. S.; Fraser, J. M.; Seaman, M.; Dunbar, D. Rat liver metabolism and toxicity of 2,2,2-trifluoroethanol. Biochem. Pharmacol. 1992, 44, 1829– 1837, DOI: 10.1016/0006-2952(92)90078-W[Crossref], [PubMed], [CAS], Google Scholar88https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXntlCquw%253D%253D&md5=637dd03291a5f959f6f5e10849b81a06Rat liver metabolism and toxicity of 2,2,2-trifluoroethanolKaminsky, Laurence S.; Fraser, Joanne M.; Seaman, Michael; Dunbar, DeborahBiochemical Pharmacology (1992), 44 (9), 1829-37CODEN: BCPCA6; ISSN:0006-2952.2,2,2-Trifluoroethanol (TFE) is a metabolite of anesthetic agents and chlorofluorocarbon alternatives. Its toxicity in rats is a consequence of its metab. to 2,2,2-trifluoroacetaldehyde (TFAld) and then to trifluoroacetic acid (TFAA). The enzymes involved in the toxic metabolic pathway have been investigated in this study. For the reaction of TFE to TFAld, the major hepatic metab. assocd. with toxicity (as assessed by pyrazole-inhibition) was NADPH dependent and occurred in the microsomes, whereas for TFAld conversion to TFAA, NADPH-dependent microsomal metab. was significant, but mitochondrial and cytosolic metab. in the presence of NADPH were also major contributors. NADPH-dependent hepatic microsomal metab. of TFE to TFAld and TFAld to TFAA was inhibited by carbon monoxide, 2-allyl-2-isopropylacetamide, SKF-525A, metyrapone, imidazole, and pyrazole, and both reactions were oxygen dependent. The metab. of TFE to TFAld was inhibited by diethyldithiocarbamate, a specific inhibitor of cytochrome P450E1, and by a monoclonal antibody to P 4502E1, whereas the metab. of TFAld was inhibited by neither agent. Ethanol pretreatment of rats enhanced the Vmax for hepatic microsomal metab. of TFE to TFAld from 5.3 to 9.7 nmol/mg protein/min, while for TFAld to TFAA, the Vmax was increased from 4.3 to 6.5 and the Km was unaffected for both reactions. Phenobarbital pretreatment of the rats did not affect any of these kinetic parameters. Coadministration of ethanol and a LD of TFE very markedly decreased the lethality. Both the lethality (LD50 0.21 to 0.44 g/kg) and the metabolic kinetic parameters [(Vmax/Km)H(Vmax/Km)D = 4.2] were affected markedly when deuterated TFE replaced TFE. In contrast, deuteration of TFAld did not affect its lethality or rates of metab., but did affect its Km. Taken together, these results indicate that P 4502E1 catalyzed toxicity-assocd. hepatic metab. of TFE to TFAld, while TFAld metab. was catalyzed by a P 450 which was not P 4502E1. The hepatic metab. of TFAld was not assocd. with its toxicity, which has been detd. previously to be assocd. with its intestinal metab.89Dowty, M. E.; Hu, G.; Hua, F.; Shilliday, F. B.; Dowty, H. V. Drug design structural alert: formation of trifluoroacetaldehyde through N-dealkylation is linked to testicular lesions in rat. Int. J. Toxicol. 2011, 30, 546– 550, DOI: 10.1177/1091581811413833[Crossref], [PubMed], [CAS], Google Scholar89https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVGktLfM&md5=30f0e43e80ba5d541b48eeee99b2e95bDrug design structural alert: formation of trifluoroacetaldehyde through N-dealkylation is linked to testicular lesions in ratDowty, Martin E.; Hu, George; Hua, Fengmei; Shilliday, F. Barclay; Dowty, Heather V.International Journal of Toxicology (2011), 30 (5), 546-550CODEN: IJTOFN; ISSN:1091-5818. (Sage Publications)In the process of drug design, it is important to consider potential structural alerts that may lead to toxicosis. This work illustrates how using trifluoroethane as a part of a novel chem. entity led to cytochrome P 450 - mediated N-dealkylation and the formation of trifluoroacetaldehyde, a known testicular toxicant, in exploratory safety studies in rats. Testicular toxicosis was noted microscopically in a dose-dependent manner as measured by testicular spermatocytic degeneration and necrosis and excessive intratubular cellular debris in the epididymis. This apparent toxic effect correlated well with the dose-dependent formation of trifluoroacetaldehyde, identified from in vitro rat liver microsome metab. studies. A similar safety study performed with an N-tetrazole substitution in place of the N-trifluoroethane showed no evidence of testicular injury, implicating further the role of trifluoroacetaldehyde in the testicular lesion obsd. These results highlight the relevance of early metabolic and safety testing in assessing potential structural alerts in drug design.90Kanduti, D.; Sterbenk, P.; Artnik, B. Fluoride: A review of use and effects on health. Mater. Sociomed. 2016, 28, 133– 137, DOI: 10.5455/msm.2016.28.133-137[Crossref], [PubMed], [CAS], Google Scholar90https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28bms1amtA%253D%253D&md5=eb0013cea0610f282fb5ff431254e8e1FLUORIDE: A REVIEW OF USE AND EFFECTS ON HEALTHKanduti Domen; Sterbenk Petra; Artnik BarbaraMateria socio-medica (2016), 28 (2), 133-7 ISSN:1512-7680.INTRODUCTION: Appropriate oral health care is fundamental for any individual's health. Dental caries is still one of the major public health problems. The most effective way of caries prevention is the use of fluoride. AIM: The aim of our research was to review the literature about fluoride toxicity and to inform physicians, dentists and public health specialists whether fluoride use is expedient and safe. METHODS: Data we used in our review were systematically searched and collected from web pages and documents published from different international institutions. RESULTS: Fluoride occurs naturally in our environment but we consume it in small amounts. Exposure can occur through dietary intake, respiration and fluoride supplements. The most important factor for fluoride presence in alimentation is fluoridated water. Methods, which led to greater fluoride exposure and lowered caries prevalence, are considered to be one of the greatest accomplishments in the 20th centurys public dental health. During pregnancy, the placenta acts as a barrier. The fluoride, therefore, crosses the placenta in low concentrations. Fluoride can be transmitted through the plasma into the mother's milk; however, the concentration is low. The most important action of fluoride is topical, when it is present in the saliva in the appropriate concentration. The most important effect of fluoride on caries incidence is through its role in the process of remineralization and demineralization of tooth enamel. Acute toxicity can occur after ingesting one or more doses of fluoride over a short time period which then leads to poisoning. Today, poisoning is mainly due to unsupervised ingestion of products for dental and oral hygiene and over-fluoridated water. CONCLUSION: Even though fluoride can be toxic in extremely high concentrations, its topical use is safe. The European Academy of Paediatric Dentistry (EAPD) recommends a preventive topical use of fluoride supplements because of their cariostatic effect.91Ullah, R.; Zafar, M. S.; Shahani, N. Potential fluoride toxicity from oral medicaments: A review. Iran. J. Basic Med. Sci. 2017, 20, 841– 848, DOI: 10.22038/IJBMS.2017.9104[Crossref], [PubMed], [CAS], Google Scholar91https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1M7mtVWjsw%253D%253D&md5=8dc1bca5b45a3fa700cd5ae62745adc9Potential fluoride toxicity from oral medicaments: A reviewUllah Rizwan; Shahani Nazish; Zafar Muhammad Sohail; Zafar Muhammad SohailIranian journal of basic medical sciences (2017), 20 (8), 841-848 ISSN:2008-3866.The beneficial effects of fluoride on human oral health are well studied. There are numerous studies demonstrating that a small amount of fluoride delivered to the oral cavity decreases the prevalence of dental decay and results in stronger teeth and bones. However, ingestion of fluoride more than the recommended limit leads to toxicity and adverse effects. In order to update our understanding of fluoride and its potential toxicity, we have described the mechanisms of fluoride metabolism, toxic effects, and management of fluoride toxicity. The main aim of this review is to highlight the potential adverse effects of fluoride overdose and poorly understood toxicity. In addition, the related clinical significance of fluoride overdose and toxicity has been discussed.92Tu, L. Q.; Wright, P. F.; Rix, C. J.; Ahokas, J. T. Is fluoroacetate-specific defluorinase a glutathione S-transferase?. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 2006, 143, 59– 66, DOI: 10.1016/j.cbpc.2005.12.003[Crossref], [PubMed], [CAS], Google Scholar92https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjtFOku7w%253D&md5=fa0621017a921fd8adb1a73e6a7fe762Is fluoroacetate-specific defluorinase a glutathione S-transferase?Tu, L. Q.; Wright, P. F. A.; Rix, C. J.; Ahokas, J. T.Comparative Biochemistry and Physiology, Part C: Toxicology & Pharmacology (2006), 143C (1), 59-66CODEN: CBPPFK; ISSN:1532-0456. (Elsevier B.V.)Fluoroacetate-specific defluorinase (FSD) is a crit. enzyme in the detoxication of fluoroacetate. This study investigated whether FSD can be classed as a glutathione S-transferase (GST) isoenzyme with a high specificity for fluoroacetate detoxication metab. The majority of FSD and GST activity, using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as GST substrates, in rat liver was cytosolic. GSTT1 specific substrate, EPNP caused a slight non-competitive inhibition of FSD activity. CDNB, a general substrate of GST isoenzyme, was a more potent non-competitive inhibitor of FSD activity. The fluoroacetate defluorination activity by GST isoenzymes was detd. in this study. The results showed that the GSTZ1C had the highest fluoroacetate defluorination activity of the various GST isoenzymes studied, while GSTA2 had a limited activity toward fluoroacetate. The human GSTZ1C recombinant protein then was purified from a human GSTZ1C cDNA clone. Our expts. showed that GSTZ1C catalyzed fluoroacetate defluorination. GSTZ1 shares many of the characteristics of FSD; however, it accounts only for 3% of the total cytosolic FSD activity. GSTZ1C based enzyme kinetic studies has low affinity for fluoroacetate. The evidence suggests that GSTZ1 may not be the major enzyme defluorinating fluoroacetate, but it does detoxify the fluoroacetate. To clarify the identity of enzymes responsible for fluoroacetate detoxication, further studies of the overall FSD activity are needed.93Jitsumori, K.; Omi, R.; Kurihara, T.; Kurata, A.; Mihara, H.; Miyahara, I.; Hirotsu, K.; Esaki, N. X-Ray crystallographic and mutational studies of fluoroacetate dehalogenase from Burkholderia sp. strain FA1. J. Bacteriol. 2009, 191, 2630– 2637, DOI: 10.1128/JB.01654-08[Crossref], [PubMed], [CAS], Google Scholar93https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXltFaku7k%253D&md5=a56bf8ff4136f279044950f5f623f301X-ray crystallographic and mutational studies of fluoroacetate dehalogenase from Burkholderia sp. strain FA1Jitsumori, Keiji; Omi, Rie; Kurihara, Tatsuo; Kurata, Atsushi; Mihara, Hisaaki; Miyahara, Ikuko; Hirotsu, Ken; Esaki, NobuyoshiJournal of Bacteriology (2009), 191 (8), 2630-2637CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)Fluoroacetate dehalogenase (FAc-DEX) catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of a carbon-fluorine bond of an aliph. compd.: the bond energy of the carbon-fluorine bond is among the highest found in natural products. The enzyme also acts on chloroacetate, although much less efficiently. We here detd. the X-ray crystal structure of the enzyme from Burkholderia sp. strain FA1 as the first exptl. detd. three-dimensional structure of fluoroacetate dehalogenase. The enzyme belongs to the α/β hydrolase superfamily and exists as a homodimer. Each subunit consists of core and cap domains. The catalytic triad, Asp104-His271-Asp128, of which Asp104 serves as the catalytic nucleophile, was found in the core domain at the domain interface. The active site was composed of Phe34, Asp104, Arg105, Arg108, Asp128, His271, and Phe272 of the core domain and Tyr147, His149, Trp150, and Tyr212 of the cap domain. An electron d. peak corresponding to a chloride ion was found in the vicinity of the Nε1 atom of Trp150 and the Nε2 atom of His149, suggesting that these are the halide ion acceptors. Site-directed replacement of each of the active-site residues, except for Trp150, by Ala caused the total loss of the activity toward fluoroacetate and chloroacetate, whereas the replacement of Trp150 caused the loss of the activity only toward fluoroacetate. An interaction between Trp150 and the fluorine atom is probably an abs. requirement for the redn. of the activation energy for the cleavage of the carbon-fluorine bond.94Kamachi, T.; Nakayama, T.; Shitamichi, O.; Jitsumori, K.; Kurihara, T.; Esaki, N.; Yoshizawa, K. The catalytic mechanism of fluoroacetate dehalogenase: a computational exploration of biological dehalogenation. Chem. - Eur. J. 2009, 15, 7394– 7403, DOI: 10.1002/chem.200801813[Crossref], [PubMed], [CAS], Google Scholar94https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXovVChsr8%253D&md5=af5089e2d9a528a30ec4a2cdd28eaa2eThe Catalytic Mechanism of Fluoroacetate Dehalogenase: A Computational Exploration of Biological DehalogenationKamachi, Takashi; Nakayama, Tomonori; Shitamichi, Osamu; Jitsumori, Keiji; Kurihara, Tatsuo; Esaki, Nobuyoshi; Yoshizawa, KazunariChemistry - A European Journal (2009), 15 (30), 7394-7403, S7394/1-S7394/56CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)The biol. dehalogenation of fluoroacetate carried out by fluoroacetate dehalogenase is discussed by using quantum mech./mol. mech. (QM/MM) calcns. for a whole-enzyme model of 10 800 atoms. Substrate fluoroacetate is anchored by a hydrogen-bonding network with water mols. and the surrounding amino acid residues of Arg105, Arg108, His149, Trp150, and Tyr212 in the active site in a similar way to haloalkane dehalogenase. Asp104 is likely to act as a nucleophile to attack the α-carbon of fluoroacetate, resulting in the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water mol. to the carbonyl carbon atom. The cleavage of the strong C-F bond is greatly facilitated by the hydrogen-bonding interactions between the leaving fluorine atom and the three amino acid residues of His149, Trp150, and Tyr212. The hydrolysis of the ester intermediate is initiated by a proton transfer from the water mol. to His271 and by the simultaneous nucleophilic attack of the water mol. The transition state and produced tetrahedral intermediate are stabilized by Asp128 and the oxyanion hole composed of Phe34 and Arg105.95Kurihara, T. A mechanistic analysis of enzymatic degradation of organohalogen compounds. Biosci., Biotechnol., Biochem. 2011, 75, 189– 198, DOI: 10.1271/bbb.100746[Crossref], [PubMed], [CAS], Google Scholar95https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjtVWrtro%253D&md5=df0a7c65a24a7c9a098d2db67a38fda3A mechanistic analysis of enzymatic degradation of organohalogen compoundsKurihara, TatsuoBioscience, Biotechnology, and Biochemistry (2011), 75 (2), 189-198CODEN: BBBIEJ; ISSN:0916-8451. (Japan Society for Bioscience, Biotechnology, and Agrochemistry)A review. Enzymes that catalyze the conversion of organohalogen compds. have been attracting a great deal of attention, partly because of their possible applications in environmental technol. and the chem. industry. The authors have studied the mechanisms of enzymic degrdn. of various org. halo acids. In the reaction of L-2-haloacid dehalogenase and fluoroacetate dehalogenase, the carboxylate group of the catalytic Asp residue nucleophilically attacks the α-C atom of the substrates to displace the halogen atom. In the reaction catalyzed by DL-2-haloacid dehalogenase, a water mol. directly attacks the substrate to displace the halogen atom. In the course of studies on the metab. of 2-chloroacrylate, the authors discovered 2 new enzymes. 2-Haloacrylate reductase catalyzes the asym. redn. of 2-haloacrylate to produce L-2-haloalkanoic acid in an NADPH-dependent manner. 2-Haloacrylate hydratase catalyzes the hydration of 2-haloacrylate to produce pyruvate. This enzyme is unique in that it catalyzes the non-redox reaction in an FADH2-dependent manner.96Chan, P. W.; Yakunin, A. F.; Edwards, E. A.; Pai, E. F. Mapping the reaction coordinates of enzymatic defluorination. J. Am. Chem. Soc. 2011, 133, 7461– 7468, DOI: 10.1021/ja200277d[ACS Full Text], [CAS], Google Scholar96https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXltVGgsb8%253D&md5=c25872673b8386d33c29e7a9b3e7ad8bMapping the Reaction Coordinates of Enzymatic DefluorinationChan, Peter W. Y.; Yakunin, Alexander F.; Edwards, Elizabeth A.; Pai, Emil F.Journal of the American Chemical Society (2011), 133 (19), 7461-7468CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The carbon-fluorine bond is the strongest covalent bond in org. chem., yet fluoroacetate dehalogenases can readily hydrolyze this bond under mild physiol. conditions. Elucidating the mol. basis of this rare biocatalytic activity will provide the fundamental chem. insights into how this formidable feat is achieved. Here, we present a series of high-resoln. (1.15-1.80 Å) crystal structures of a fluoroacetate dehalogenase, capturing snapshots along the defluorination reaction: the free enzyme, enzyme-fluoroacetate Michaelis complex, glycolyl-enzyme covalent intermediate, and enzyme-product complex. We demonstrate that enzymic defluorination requires a halide pocket that not only supplies three hydrogen bonds to stabilize the fluoride ion but also is finely tailored for the smaller fluorine halogen atom to establish selectivity toward fluorinated substrates. We have further uncovered dynamics near the active site which may play pivotal roles in enzymic defluorination. These findings may ultimately lead to the development of novel defluorinases that will enable the biotransformation of more complex fluorinated org. compds., which in turn will assist the synthesis, detoxification, biodegrdn., disposal, recycling, and regulatory strategies for the growing markets of organofluorines across major industrial sectors.97Miranda-Rojas, S.; Toro-Labbe, A. Mechanistic insights into the dehalogenation reaction of fluoroacetate/fluoroacetic acid. J. Chem. Phys. 2015, 142, 194301, DOI: 10.1063/1.4920946[Crossref], [PubMed], [CAS], Google Scholar97https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXoslOrtLc%253D&md5=e335eff76c2b5edf972eb445c484d5c9Mechanistic insights into the dehalogenation reaction of fluoroacetate/fluoroacetic acidMiranda-Rojas, Sebastian; Toro-Labbe, AlejandroJournal of Chemical Physics (2015), 142 (19), 194301/1-194301/9CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)Fluoroacetate is a toxic compd. whose environmental accumulation may represent an important contamination problem, its elimination is therefore a challenging issue. Fluoroacetate dehalogenase catalyzes its degrdn. through a two step process initiated by an SN2 reaction in which the aspartate residue performs a nucleophilic attack on the carbon bonded to the fluorine; the second step is hydrolysis that releases the product as glycolate. In this paper, we present a study based on d. functional theory calcns. of the SN2 initiation reaction modeled through the interaction between the substrate and the propionate anion as the nucleophile. Results are analyzed within the framework of the reaction force and using the reaction electronic flux to identify and characterize the electronic activity that drives the reaction. Our results reveal that the selective protonation of the substrate catalyzes the reaction by decreasing the resistance of the structural and electronic reorganization needed to reach the transition state. Finally, the reaction energy is modulated by the degree of stabilization of the fluoride anion formed after the SN2 reaction. In this way, a site-induced partial protonation acts as a chem. switch in a key process that dets. the output of the reaction. (c) 2015 American Institute of Physics.98Li, Y.; Zhang, R.; Du, L.; Zhang, Q.; Wang, W. Catalytic mechanism of C–F bond cleavage: insights from QM/MM analysis of fluoroacetate dehalogenase. Catal. Sci. Technol. 2016, 6, 73– 80, DOI: 10.1039/C5CY00777A[Crossref], [CAS], Google Scholar98https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFShtr3E&md5=ad0a17050e722c1e117dd21015fbac23Catalytic mechanism of C-F bond cleavage: insights from QM/MM analysis of fluoroacetate dehalogenaseLi, Yanwei; Zhang, Ruiming; Du, Likai; Zhang, Qingzhu; Wang, WenxingCatalysis Science & Technology (2016), 6 (1), 73-80CODEN: CSTAGD; ISSN:2044-4753. (Royal Society of Chemistry)The catalytic mechanisms of fluoroacetate dehalogenase (FAcD) toward substrates fluoroacetate and chloroacetate were studied by a combined quantum mechanics/mol. mechanics (QM/MM) method. There are twenty snapshots considered for each of the three individual systems. By analyzing multiple independent snapshots, pos. or neg. relationships between energy barriers and structural parameters in defluorination and dechlorination processes were established. We have also shown that conformational variations may cause enzymic preference differences toward competitive pathways. Besides residues Arg111, Arg114, His155, Trp156, and Tyr219, the importance of residues His109, Asp134, Lys181, and His280 during the defluorination process were also highlighted through electrostatic anal. These results may provide clues for designing new biomimetic catalysts toward degrdn. of fluorinated compds.99Wang, J. B.; Ilie, A.; Yuan, S.; Reetz, M. T. Investigating substrate scope and enantioselectivity of a defluorinase by a stereochemical probe. J. Am. Chem. Soc. 2017, 139, 11241– 11247, DOI: 10.1021/jacs.7b06019[ACS Full Text], [CAS], Google Scholar99https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1eks7rE&md5=ad6fc8676e19c3964d6ccf9426527ab1Investigating Substrate Scope and Enantioselectivity of a Defluorinase by a Stereochemical ProbeWang, Jian-bo; Ilie, Adriana; Yuan, Shuguang; Reetz, Manfred T.Journal of the American Chemical Society (2017), 139 (32), 11241-11247CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The possibility of a double Walden inversion mechanism of the defluorinase, fluoroacetate dehalogenase FAcD (RPA1163), was studied by subjecting rac-2-fluoro-2-phenylacetic acid to the defluorination process. This stereochem. probe led to inversion of configuration in a kinetic resoln. with an extremely high selectivity factor (E = >500), showing that the classical mechanism involving SN2 reaction by Asp-110 pertained. The high preference for the (S)-substrate was of synthetic value. The wide substrate scope of RPA1163 in such hydrolytic kinetic resolns. can be expected because the reaction of the even more sterically demanding rac-2-fluoro-2-benzylacetic acid proceeded similarly. Substrate acceptance and stereoselectivity were explained by extensive mol. modeling and mol. dynamics simulation computations. These computations were also applied to fluoroacetic acid itself, leading to further insights.100Tong, Z.; Board, P. G.; Anders, M. W. Glutathione transferase zeta-catalyzed biotransformation of dichloroacetic acid and other α-haloacids. Chem. Res. Toxicol. 1998, 11, 1332– 1338, DOI: 10.1021/tx980144f[ACS Full Text], [CAS], Google Scholar100https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXmsVWjtLk%253D&md5=03d205a0bf95e6836799c6321893417dGlutathione Transferase Zeta-Catalyzed Biotransformation of Dichloroacetic Acid and Other α-HaloacidsTong, Zeen; Board, Philip G.; Anders, M. W.Chemical Research in Toxicology (1998), 11 (11), 1332-1338CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)Dichloroacetic acid (DCA) is a common drinking-water contaminant, is hepatocarcinogenic in rats and mice, and is a therapeutic agent used clin. in the management of lactic acidosis. Recent studies show that glutathione transferase Zeta (GSTZ) catalyzes the oxygenation of DCA to glyoxylic acid (1998). In the present studies, the substrate selectivity of GSTZ, the kinetics of DCA metab., and the fate of DCA and glutathione were investigated. The results showed that GSTZ catalyzed the oxygenation of bromochloro-, bromofluoro-, chlorofluoro-, dibromo-, and dichloroacetic acid, but not difluoroacetic acid, to glyoxylic acid. GSTZ also catalyzed the biotransformation of fluoroacetic acid to S-(carboxymethyl)glutathione, and of (R,S)-2-bromopropionic acid, (R)-, (S)-, and (R,S)-2-chloropropionic acid, and (R,S)-2-iodopropionic acid, but not (R,S)-2-fluoropropionic acid, to S-(α-methylcarboxymethyl)glutathione; and of 2,2-dichloropropionic acid to pyruvate. No biotransformation of 3,3-dichloropropionic acid was detected, and no GSTZ-catalyzed fluoride release from Et fluoroacetate and fluoroacetamide was obsd. The relative rates of DCA biotransformation by hepatic cytosol were mouse > rat > human. Immunoblotting showed the presence of GSTZ in mouse, rat, and human liver cytosol. 13C NMR spectroscopic studies showed that [2-13C]glyoxylic acid was the only observable, stable metabolite of [2-13C]DCA. Also, glutathione was required, but was neither consumed nor oxidized to glutathione disulfide, during the oxygenation of DCA to glyoxylic acid. These results are consistent with a reaction mechanism that involves displacement of chloride from DCA by glutathione to afford S-(α-chlorocarboxymethyl)glutathione, which may undergo hydrolysis to give the hemithioacetal S-(α-hydroxycarboxymethyl)glutathione. Elimination of glutathione from the hemithioacetal would give glyoxylic acid.101Soiefer, A. I.; Kostyniak, P. J. Purification of a fluoroacetate-specific defluorinase from mouse liver cytosol. J. Biol. Chem. 1984, 259, 10787– 10792[PubMed], [CAS], Google Scholar101https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXmt1Ggu7k%253D&md5=5202bbd98e7f723aab7dd5950389ecf7Purification of a fluoroacetate-specific defluorinase from mouse liver cytosolSoiefer, Andrew I.; Kostyniak, Paul J.Journal of Biological Chemistry (1984), 259 (17), 10787-92CODEN: JBCHA3; ISSN:0021-9258.Fluoroacetate-specific defluorinase (I), an enzyme which catalyzes the release of F- from the rodenticide, fluoroacetate, was purified 347-fold from mouse liver cytosol and shown to be distinct from multiple cationic and anionic glutathione S-transferase isoenzymes. Fluoroacetate-specific I was obtained at a final specific activity of 659 nmol of F-/min/mg protein and was prepd. in an overall yield of 12%. The pI of this hepatic enzyme was acidic, at pH 6.4, as detd. by column chromatofocusing. The mol. wt. of the active species was estd. at 41,000 and SDS-polyacrylamide gels of purified I demonstrated a predominant subunit of mol. wt. 27,000. Chromatofocusing completely partitioned fluoroacetate-specific I from 2 sep. peaks of murine anionic glutathione S-transferase activity. Rabbit antibodies prepd. against purified hepatic I quant. pptd. native I from mouse and rat liver, but were unable to immunoppt. cationic or anionic glutathione S-transferase enzymes from the same prepn. The evidence presented suggests that fluoroacetate-specific I and glutathione S-transferase activities are catalyzed by sep. proteins present in the cytosol of mouse liver.102Kostyniak, P. J.; Soiefer, A. I. The role of fluoroacetate-specific dehalogenase and glutathione transferase in the metabolism of fluoroacetamide and 2,4-dinitrofluorobenzene. Toxicol. Lett. 1984, 22, 217– 222, DOI: 10.1016/0378-4274(84)90069-9[Crossref], [PubMed], [CAS], Google Scholar102https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXmt1Ciuro%253D&md5=e4eafada4752417c0223d81d968c16acThe role of fluoroacetate-specific dehalogenase and glutathione transferase in the metabolism of fluoroacetamide and 2,4-dinitrofluorobenzeneKostyniak, P. J.; Soiefer, A. I.Toxicology Letters (1984), 22 (2), 217-22CODEN: TOLED5; ISSN:0378-4274.2,4-Dinitrofluorobenzene (DNFB)(I) [70-34-8] reacts with glutathione [70-18-8] to form a stable product similar to that formed with the model glutathione S-transferase (GST) [50812-37-8] substrate, 1-chloro-2,4-dinitrobenzene (CDNB) [97-00-7]. DNFB is ∼140 times as reactive as CDNB in this chem. reaction. The enzymic defluorination of DNFB also proceeds at a more rapid rate than CDNB in the GST assay. Fluoroacetamide (FAM) [640-19-7], like fluoroacetate (FAC) [144-49-0] undergoes no discernable chem. defluorination. Its enzymic defluorination is ∼10% of that obsd. for FAC and only 0.2% of the rate for DNFB. An antibody raised to the fluoroacetate-specific dehalogenase (FSD) [37289-40-0] pptd. FAC and FAM defluorinating activity, but had no effect on CDNB or DNFB activity. Thus, DNFB is metabolized by the GST while FAM is metabolized by the FSD.103Tecle, B.; Casida, J. E. Enzymatic defluorination and metabolism of fluoroacetate, fluoroacetamide, fluoroethanol, and (−)-erythro-fluorocitrate in rats and mice examined by 19F and 13C NMR. Chem. Res. Toxicol. 1989, 2, 429– 435, DOI: 10.1021/tx00012a012[ACS Full Text], [CAS], Google Scholar103https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXmtFSgsr4%253D&md5=f38dbfdf56eb267c89a237b4c068583fEnzymatic defluorination and metabolism of fluoroacetate, fluoroacetamide, fluoroethanol, and (-)-erythro-fluorocitrate in rats and mice examined by fluorine-19 and carbon-13 NMRTecle, Berhane; Casida, John E.Chemical Research in Toxicology (1989), 2 (6), 429-35CODEN: CRTOEC; ISSN:0893-228X.Fluoroacetate administered i.p. to rats and mice is defluorinated to give fluoride ion evident in urine and kidney by 19F NMR. The use of [2-13C]- and [1,2-14C]fluoroacetate, prepd. from isotopically labeled glycine, combined with 13C NMR and TLC radioautog., resp., reveals a complex mixt. of urinary metabolites including an S-(carboxymethyl) conjugate complex in rats and mice and sulfoxidn. products thereof in rats. Direct 13C NMR examn. of the bile following treatment with [2-13C]fluoroacetate shows the presence of S-(carboxymethyl)glutathione or a related conjugate and an O-conjugate of fluoroacetate. Incubation of [13C]fluoroacetate with rat and mouse liver cytosol involves formation of S-[([13C]carboxymethyl)glutathione and fluoride ion. Fluorocitrate is also detected by 19F NMR examn. of fluoroacetate incubations with mouse liver cytosol. Fluoroacetamide administered i.p. to rats and mice yields urinary fluoride ion formed via fluoroacetate which is liberated on hydrolysis by an organophosphate-sensitive amidase. 19F NMR chem. shifts of other metabolites of fluoroacetamide are consistent with fluoroacetohydroxamic acid in the liver of mice and fluorocitrate in the urine of rats. Fluoroethanol gives urinary fluoroacetate and fluoride ion in rats and mice and is converted to fluoroacetaldehyde by mouse and rat liver microsomes. (-) And (+)-erythro-fluorocitrates administered i.p. to rats yield mostly the parent compds. in urine at 6 h with increasing amts. of fluoride ion thereafter. 19F NMR establishes that rat and mouse liver cytosols defluorinate (-)-, but not (+)-erythro-fluorocitrate and pig heart aconitase also defluorinates (-)-erythro-fluorocitrate. Metabolic defluorination of fluoroacetate and its progenitors, fluoroacetamide and fluoroethanol, is therefore attributable to both conjugation of fluoroacetate with GSH and conversion to (-)-erythro-fluorocitrate, which is both an inhibitor of and a substrate for aconitase. 13C NMR spectra of urine of rats and mice poisoned with fluoroacetate or (-)-erythro-fluorocitrate show elevated citrate and glucose and diminished urea consistent with disruptions in the tricarboxylic acid cycle and NH3 metab.104Kim, K.-H.; Shon, Z.-H.; Nguyen, H. T.; Jeon, E.-C. A review of major chlorofluorocarbons and their halocarbon alternatives in the air. Atmos. Environ. 2011, 45, 1369– 1382, DOI: 10.1016/j.atmosenv.2010.12.029[Crossref], [CAS], Google Scholar104https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFeis7Y%253D&md5=2e83d2921958c8033e2f28df1934949eA review of major chlorofluorocarbons and their halocarbon alternatives in the airKim, Ki-Hyun; Shon, Zang-Ho; Nguyen, Hang Thi; Jeon, Eui-ChanAtmospheric Environment (2011), 45 (7), 1369-1382CODEN: AENVEQ; ISSN:1352-2310. (Elsevier Ltd.)A review; to establish a proper regulation strategy on the emissions of major halocarbons including chlorofluorocarbons (CFCs), carbon tetrachloride (CCl4), hydrochlorofluorocarbons (HCFCs) and hydrofluorocarbons (HFCs), it is essential to assess their environmental behavior in relation to social and technol. changes for their control. The prodn. and release of CFCs and CCl4 increased rapidly to peak between the 1970s and 1980s and then decreased dramatically from the end of the 1980s in good accordance with the phase out schedules set by the Montreal Protocol. Both HCFCs (e.g., HCFC-124, HCFC-141b, and HCFC-142b) and HFCs (e.g., HFC-134a) have been introduced as CFCs alternatives between the late 1980s and early 1990s. However, these alternatives have already been or will be scheduled to be phased out because of their involvement in ozone destruction and global warming. In light of all the complexities assocd. with the global chem. of CFCs and their alternatives, this paper provides an overview of their prodn. and emission trends, their relationship with the ozone depletion phenomenon, the chem. regulating their removal processes, and their distribution patterns with diverse temporal and spatial scales.105Anders, M. W. Metabolism and toxicity of hydrochlorofluorocarbons: current knowledge and needs for the future. Environ. Health Perspect. 1991, 96, 185– 191, DOI: 10.1289/ehp.9196185[Crossref], [PubMed], [CAS], Google Scholar105https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xkt1Wms7k%253D&md5=d2f4f940f2d5464ed79764e06a8059cbMetabolism and toxicity of hydrochlorofluorocarbons: current knowledge and needs for the futureAnders, M. W.Environmental Health Perspectives (1991), 96 (), 185-91CODEN: EVHPAZ; ISSN:0091-6765.A review with 63 refs. Hydrochlorofluorocarbons (HCFCs) are being developed as replacements for chlorofluorocarbons (CFCs) that deplete stratospheric ozone. The depletion of stratospheric ozone may increase the intensity of UV radiation at the earth's surface, which may be assocd. with global, adverse human health effects. The greater tropospheric lability of HCFCs, which is due to the presence of C-H bonds, reduces HCFC migration to the stratosphere; HCFCs should, therefore, cause less depletion of stratospheric ozone than CFCs. HCFCs under development include HCFC-22 (chlorodifluoromethane), HCFC-123 (2,2-dichloro-1,1,1-trifluoroethane), HCFC-132b (1,2-dichloro-1,1-difluoroethane), HCFC-134a (1,1,1,2-tetrafluoroethane), HCFC-141b (1,1-dichloro-1-fluoroethane, and HCFC-142b (1-chloro-1,1-difluoroethane). With the exception of HCFC-22, which is already in use, the metab. and toxicity of HCFCs have not been studied in detail. By analogy to chlorinated ethanes, predictions can be made about the possible metab. of HCFCs, but there are insufficient data available to predict rates of metab. Although most HCFCs appear to show low acute toxicity, some HCFCs are mutagenic in the Ames test. Hence, future research on HCFCs should include studies on the in vivo and in vitro metab. of HCFCs as well as on their toxicity in in vivo and in vitro systems.106Urban, G.; Dekant, W. Metabolism of 1,1-dichloro-2,2,2-trifluoroethane in rats. Xenobiotica 1994, 24, 881– 892, DOI: 10.3109/00498259409043287[Crossref], [PubMed], [CAS], Google Scholar106https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXmsVynsLw%253D&md5=42c9c0f6a5266d7ca1de4b1947502296Metabolism of 1,1-dichloro-2,2,2-trifluoroethane in ratsUrban, G.; Dekant, W.Xenobiotica (1994), 24 (9), 881-92CODEN: XENOBH; ISSN:0049-8254.1,1-Dichloro-2,2,2-trifluoro-[2-14C]-ethane (HCFC-123) is a chlorofluorohydrocarbon with potential widespread use and assocd. human exposure. As a part of the toxicol. evaluation of HCFC-123, its metab. was studied in rodents in a closed recirculating exposure system. Two male rats were individually exposed for 6 h. Excretion of radioactivity was monitored for 48 h after the start of the exposure. Of the radioactivity introduced into the chamber, 14% was recovered in urine within the period of observation. Excretion of metabolites in the urine was very slow. Trifluoroacetic acid was the major metabolite of HCFC-123 and N-trifluoroacetyl-2-aminoethanol and N-acetyl-S-(2,2-dichloro-1,1-difluoroethyl)-L-cysteine were identified as minor urinary metabolites of HCFC-123. Forty-eight hours after the start of the exposure, covalent binding of radioactive metabolites to protein was highest in liver followed by kidney and lung. Covalent binding above background levels was not obsd. in pancreas and testis, the target organs of HCFC-123 tumorigenicity. These results suggest that the biotransformation of HCFC-123 in rodents follows a pathway identical to those of the extensively studied structural analog halothane.107Urban, G.; Speerschneider, P.; Dekant, W. Metabolism of the chlorofluorocarbon substitute 1,1-dichloro-2,2,2-trifluoroethane by rat and human liver microsomes: the role of cytochrome P450 2E1. Chem. Res. Toxicol. 1994, 7, 170– 176, DOI: 10.1021/tx00038a009[ACS Full Text], [CAS], Google Scholar107https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXhvVCrsL8%253D&md5=e59b32f3e2b9891d2c6df95d05e5201aMetabolism of the Chlorofluorocarbon Substitute 1,1-Dichloro-2,2,2-trifluoroethane by Rat and Human Liver Microsomes: The Role of Cytochrome P450 2E1Urban, Gudrun; Speerschneider, Petra; Dekant, WolfgangChemical Research in Toxicology (1994), 7 (2), 170-6CODEN: CRTOEC; ISSN:0893-228X.1,1-Dichloro-2,2,2-trifluoroethane (HCFC-123) has been developed as a substitute for ozone-depleting chlorofluorocarbons. The authors compared the metab. of HCFC-123 and its analog halothane in rat and human liver microsomes. 19F-NMR studies showed that trifluoroacetic acid is a major metabolite of HCFC-123. Besides trifluoroacetic acid, chlorodifluoroacetic acid and inorg. fluoride were identified as products of the enzymic oxidn. of HCFC-123 in rat and human liver microsomes by 19F-NMR and mass spectrometry. These metabolites were not detected in incubations with halothane. HCFC-123 and halothane were transformed by liver microsomes from untreated rats at low rates. Microsomes from ethanol- and pyridine-treated rats metabolized both HCFC-123 and halothane at much higher rates. These microsomes also exhibited high rates of p-nitrophenol oxidn. P-Nitrophenol is a model substrate mainly oxidized by P 450 2E1 to p-nitrocatechol. Samples of human liver microsomes showed considerable differences in the extent of HCFC-123, p-nitrophenol oxidn., and chlorzoxazone hydroxylation. In human liver microsomes, rabbit anti-rat P 450 2E1 IgG recognized a single protein band corresponding in apparent mol. wt. to human P 450 2E1. Immunoblot anal. revealed considerable heterogenity in the P 450 2E1 protein content of the human liver samples. Trifluoroacetic acid formation from HCFC-123 and halothane and p-nitrocatechol formation from p-nitrophenol were significantly reduced by the P 450 2E1 inhibitor diethyldithiocarbamate. P-Nitrophenol also inhibited halothane and HCFC-123 oxidn. in both rat and human liver microsomes. Moreover, the rates of trifluoroacetic acid formation from HCFC-123 and halothane correlated well with the ability of rat and human liver microsomes to oxidize p-nitrophenol and chlorzoxazone and the amt. of P 450 2E1 protein in liver microsomes detd. by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot anal. These data indicate that cytochrome P 450 2E1 plays a major role in the metab. of HCFC-123 and halothane in vitro.108Olson, M. J.; Johnson, J. T.; O’Gara, J. F.; Surbrook, S. E., Jr. Metabolism in vivo and in vitro of the refrigerant substitute 1,1,1,2-tetrafluoro-2-chloroethane. Drug Metab. Dispos. 1991, 19, 1004– 1011[PubMed], [CAS], Google Scholar108https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXmt1Glu7c%253D&md5=edf8bcea5ee06045607c6e6c47a36383Metabolism in vivo and in vitro of the refrigerant substitute 1,1,1,2-tetrafluoro-2-chloroethaneOlson, Michael J.; Johnson, John T.; O'Gara, John F.; Surbrook, Stephen E., Jr.Drug Metabolism and Disposition (1991), 19 (5), 1004-11CODEN: DMDSAI; ISSN:0090-9556.Ternary mixts. of hydrochlorofluorocarbons and hydrofluorocarbons are being evaluated as refrigerant substitutes for dichlorodifluoromethane, which is to be banned from further prodn. in 2000. A priori consideration of the similarity between 1,1,1,2-tetrafluoro-2-chloroethane (HCFC-124), a primary component of candidate refrigerant blends, and halothane suggests that metab. of HCFC-124 might proceed via reactive intermediates. The data show that rats exposed for 2 h to ∼10,000 ppm HCFC-124 excreted both inorg. fluoride (F-) and trifluoroacetic acid (TFA), identified by 9F-NMR, in the urine. Likewise, microsomes produced F- and TFA from HCFC-124 in an NADPH-dependent, CO-inhibited, aerobic reaction. Treatment of rats with pyridine caused about a 20-fold increase in aerobic microsomal metab. (F- release) of HCFC-124, while the rate of defluorination was slightly decreased by phenobarbital administration. An antibody to cytochrome P 450 IIE1 inhibited more than 90% of HCFC-124 metab. in pyridine-induced prepns. Defluorination of HCFC-124 by microsomes also occurred under conditions of greatly reduced oxygen tension, demonstrating that this halocarbon can be reductively metabolized. Moreover, heat-inactivated, NADPH-reduced microsomes liberated F- and a fluorinated org. product, although not TFA, from HCFC-124. Formation of TFA and F- as products of oxidative HCFC-124 metab. support the hypothesis that trifluoroacetyl fluoride is formed as an intermediate. Trifluoroacetyl halides are known to adduct tissue proteins. The reductive metab. of HCFC-124, by analogy to halothane, may produce a radical (•CHFCF) capable of biol. interactions. Although the authors offer evidence for the cytochrome P 450-dependent metab. of HCFC-124 both in vivo and in vitro, it is, at present, unknown whether the rate of HCFC-124 metab. in vivo is sufficient to result in trifluoroacetylation of tissue proteins or other protoxic events mediated by reactive intermediates.109Yin, H.; Jones, J. P.; Anders, M. W. Metabolism of 1-fluoro-1,1,2-trichloroethane, 1,2-dichloro-1,1-difluoroethane, and 1,1,1-trifluoro-2-chloroethane. Chem. Res. Toxicol. 1995, 8, 262– 268, DOI: 10.1021/tx00044a012[ACS Full Text], [CAS], Google Scholar109https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXjvVSksrk%253D&md5=f832e3f587d90005f9736302d2dfeddaMetabolism of 1-Fluoro-1,1,2-trichloroethane, 1,2-Dichloro-1,1-difluoroethane, and 1,1,1-Trifluoro-2-chloroethaneYin, Hequn; Jones, Jeffrey P.; Anders, M. W.Chemical Research in Toxicology (1995), 8 (2), 262-8CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)1-Fluoro-1,1,2-trichloroethane (HCFC-131a), 1,2-dichloro-1,1-difluoroethane (HCFC-132b), and 1,1,1-trifluoro-2-chloroethane (HCFC-133a) were chosen as models for comparative metab. studies on 1,1,1,2-tetrahaloethanes, which are under consideration as replacements for ozone-depleting chlorofluorocarbons (CFCs). Male Fischer 344 rats were given 10 mmol/kg i.p. HCFC-131a or HCFC-132b or exposed by inhalation to 1% HCFC-133a for 2 h. Urine collected in the first 24 h after exposure was analyzed by 19F NMR and GC/MS and with a fluoride-selective ion electrode for the formation of fluorine-contg. metabolites. Metabolites of HCFC-131a included 2,2-dichloro-2-fluoroethyl glucuronide, 2,2-dichloro-2-fluoroethyl sulfate, dichlorofluoroacetic acid, and inorg. fluoride. Metabolites of HCFC-132b were characterized as 2-chloro-2,2-difluoroethyl glucuronide, 2-chloro-2,2-difluoroethyl sulfate, chlorodifluoroacetic acid, chlorodifluoroacetaldehyde hydrate, chlorodifluoroacetaldehyde-urea adduct, and inorg. fluoride. HCFC-133a was metabolized to 2,2,2-trifluoroethyl glucuronide, trifluoroacetic acid, trifluoroacetaldehyde hydrate, trifluoroacetaldehyde-urea adduct, inorg. fluoride, and a minor, unidentified metabolite. With HCFC-131a and HCFC-132b, glucuronide conjugates of 2,2,2-trihaloethanols were the major urinary metabolites, whereas with HCFC-133a, a trifluoroacetaldehyde-urea adduct was the major urinary metabolite. Anal. of metabolite distribution in vivo indicated that aldehydic metabolites increased as fluorine substitution increased in the order HCFC-131a < HCFC-132b < HCFC-133a. With NADPH-fortified rat liver microsomes, HCFC-133a and HCFC-132b were biotransformed to trifluoroacetaldehyde and chlorodifluoroacetaldehyde, resp., whereas HCFC-131a was converted to dichlorofluoroacetic acid. No covalently bound metabolites of HCFC-131a and HCFC-133a metabolites were detected by 19F NMR spectroscopy. The nature of the identified org. fluorine-contg. metabolites indicates that cytochrome P 450-dependent oxidn. predominates in the metab. of these 1,1,1,2-tetrahaloethanes. The generation of fluoride from the fluorodihalomethyl group (-CFX2) apparently arises from a sep. dehalogenation pathway.110Harris, J. W.; Anders, M. W. Metabolism of the hydrochlorofluorocarbon 1,2-dichloro-1,1-difluoroethane. Chem. Res. Toxicol. 1991, 4, 180– 186, DOI: 10.1021/tx00020a009[ACS Full Text], [CAS], Google Scholar110https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXhtFKmsrc%253D&md5=6aaa6391e895b4178d6acc9baf150325Metabolism of the hydrochlorofluorocarbon 1,2-dichloro-1,1-difluoroethaneHarris, James W.; Anders, M. W.Chemical Research in Toxicology (1991), 4 (2), 180-6CODEN: CRTOEC; ISSN:0893-228X.Male rats were given 10 mmol/kg 1,2-dichloro-1,1-difluoroethane (HCFC-132b) dissolved in corn oil by i.p. injection. An NMR assay for covalent binding of HCFC-132b metabolites to liver proteins was neg., whereas binding was obsd. in halothane-treated rats. Total urinary metabolites excreted by rats given HCFC-132b during the first 24 h amounted to 1.8% of the injected dose, as detd. by 19F NMR. During the first 6 h, metabolites of HCFC-132b corresponding to 2-chloro-2,2-difluoroethyl glucuronide, unknown metabolite A, chlorodifluoroacetic acid, and chlorodifluoroacetaldehyde hydrate [both free and conjugated (unknown metabolite B)] were excreted in urine in the approx. ratio 100 : 9 : 3 : 7, resp. Metabolite A is apparently an O-conjugate of 2-chloro-2,2-difluoroethanol; unconjugated 2-chloro-2,2-difluoroethanol was not detected in urine. The 19F NMR spectrum of metabolite B indicates the formation of a hemiacetal of chlorodifluoroacetaldehyde. Repeated exposure of rats to HCFC-132b significantly increased both the rate of chlorodifluoroacetic acid excretion and the relative fraction of the HCFC-132b dose excreted as chlorodifluoroacetic acid in urine. Incubation of HCFC-132b with rat hepatic microsomes yielded chlorodifluoroacetaldehyde hydrate as the only fluorinated product. The in vitro metab. of HCFC-132b was increased in microsomes from pyridine-treated rats as compared with control rats and HCFC-132b metab. was inhibited by p-nitrophenol, indicating that the cytochrome P 450 isoform IIE1 is largely responsible for the initial hydroxylation of HCFC-132b.111Rusch, G. M.; Coombs, D.; Hardy, C. The acute, genetic, developmental, and inhalation toxicology of 1,1,1,3,3-pentafluoropropane (HFC 245fa). Toxicol. Sci. 1999, 52, 289– 301, DOI: 10.1093/toxsci/52.2.289[Crossref], [PubMed], [CAS], Google Scholar111https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXhtF2itA%253D%253D&md5=0955f74d095eb9344c1ebf0ab27f935cThe acute, genetic, developmental, and inhalation toxicology of 1,1,1,3,3-pentafluoropropane (HFC 245fa)Rusch, George M.; Coombs, Derek; Hardy, ColinToxicological Sciences (1999), 52 (2), 289-301CODEN: TOSCF2; ISSN:1096-6080. (Oxford University Press)1,1,1,3,3-Pentafluoropropane (HFC 245fa) is a volatile, low boiling liq. Tt was inactive in a reverse mutation (Ames) assay using 5 strains of Salmonella typhimurium and 1 strain of Escherichia coli. It was also inactive in an in vivo mouse micronucleus assay with exposures of 101,000 ppm. In a chromosome aberration study with human lymphocytes, some activity was seen when cell cultures were exposed to atmospheres of 30% vol./vol. or higher for 24 h without metabolic activation. No activity was seen in assays using <30% vol./vol. or exposure times of less than 24 h. No activity was seen in the presence of metabolic activation even with exposures of 70%. It was not toxic by the dermal route. There was no mortality or significant signs of toxicity when rats and mice were given 4 h exposures to levels of 203,000 ppm or 101,000 ppm of HFC 245fa, resp. In a cardiac sensitization study with dogs involving i.v. administration of epinephrine, the no obsd. effect level (NOEL) was 34,000 ppm and the threshold for a response was 44,000 ppm. In a rat inhalation, developmental toxicity study, a slight redn. in pup wt. was seen at 50,000 ppm, but not at 10,000 ppm. There were no developmental effects at any level. A series of 3 inhalation toxicity studies were conducted. All involved daily 6 h exposures ≤50,000 ppm. The 1st study involved 14 consecutive snout-only exposures. There were no treatment-related effects on body wt., survival, or histol. parameters. BUN, GPT, and GOT levels frequently were elevated compared to controls, whereas cholesterol levels tended to be lower. The 2nd study involved 28 consecutive whole-body exposures. Again, there were no treatment related effects on body wt., survival, or histol. parameters. Urine vol. was increased. Increases were also seen in several red blood cell parameters. These may be related to partial dehydration. Increases were seen in BUN levels and alk. phosphatase (AP), GPT, GOT, and CPK activities, primarily in rats exposed at 10,000 and 50,000 ppm. Urinary fluoride levels were also elevated in an exposure- related pattern. In the 3rd study, whole-body exposures were conducted 5 days per wk for 13 wk. There were no treatment-related effects on survival, clin. observations, body wt. gain, or food consumption. Urine vols. were increased, urinary fluoride levels were elevated, and increases were seen in red blood cell counts, and related parameters and increases were seen in AP, GOT, GPT, and CPK activities. These effects were seen in the 10,000 and 50,000 ppm exposure level groups. Histopathol. examn. did not show any effects on the kidney, liver, or lungs. There was an increased incidence of myocarditis in all animals exposed at 50,000 ppm and the majority exposed at 10,000 ppm. It was described as mild. Based on these findings, 2000 ppm appears to be a no obsd. adverse effect level.112Bayer, T.; Amberg, A.; Bertermann, R.; Rusch, G. M.; Anders, M. W.; Dekant, W. Biotransformation of 1,1,1,3,3-pentafluoropropane (HFC-245fa). Chem. Res. Toxicol. 2002, 15, 723– 733, DOI: 10.1021/tx025505c[ACS Full Text], [CAS], Google Scholar112https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XivFWitb0%253D&md5=9fb4489e1de801b3cb9a6183168312d9Biotransformation of 1,1,1,3,3-Pentafluoropropane (HFC-245fa)Bayer, Tanja; Amberg, Alexander; Bertermann, Ruediger; Rusch, George M.; Anders, M. W.; Dekant, WolfgangChemical Research in Toxicology (2002), 15 (5), 723-733CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)1,1,1,3,3-Pentafluoropropane (HFC-245fa) is being developed as a CFC substitute. 1,1,1,3,3-Pentafluoropropane has a low potential for toxicity: the only remarkable toxic effect seen in rats after inhalation exposure to 1,1,1,3,3-pentafluoropropane in concns. of up to 50,000 ppm for 90 days was an increased incidence of diffuse myocarditis. To elucidate the possible role of biotransformation in 1,1,1,3,3-pentafluoropropane-induced cardiotoxicity, the biotransformation of 1,1,1,3,3-pentafluoropropane was investigated in rats after inhalation exposure and in rat and human liver microsomes. Male and female rats were exposed by inhalation to 50 000, 10 000, and 2000 ppm 1,1,1,3,3-pentafluoropropane for 6 h, urine was collected for 72 h, and metabolites excreted were identified by 19F NMR spectroscopy and quantified by GC/MS. Trifluoroacetic acid and inorg. fluoride were identified as major urinary metabolites of 1,1,1,3,3-pentafluoropropane; 3,3,3-trifluoropropanoic acid and 1,1,1,3,3-pentafluoropropane-2-ol were minor metabolites. The extent of 1,1,1,3,3-pentafluoropropane biotransformation after inhalation was dependent on exposure concns. Neither 3,3,3-trifluoropropanoic acid nor 3,3,3-trifluoropyruvic acid were metabolized to trifluoroacetic acid in vitro or in rats. In rat and human liver microsomes, 1,1,1,3,3-pentafluoropropane was biotransformed by a cytochrome P 450-dependent reaction to trifluoroacetic acid and 3,3,3-trifluoropropanoic acid. Rates of trifluoroacetic acid formation were 99.2±20.5 pmol (mg of protein)-1 min-1 and of 3,3,3-trifluoropropanoic acid formation were 17.5±4.0 pmol (mg of protein)-1 min-1 in liver microsomes from male rats. In human liver microsomes, rates of trifluoroacetic acid formation ranged from 0 to 11.6 pmol (mg of protein)-1 min-1, and rates of 3,3,3-trifluoropropanoic acid formation ranged from 0.7 to 7.6 pmol (mg of protein)-1 min-1. The results show that 1,1,1,3,3-pentafluoropropane is metabolized at low rates in vivo and in vitro. The toxic effects of 1,1,1,3,3-pentafluoropropane may be assocd. with the formation of the minor metabolite 3,3,3-trifluoropropanoic acid, which is highly toxic in rats.113Mitsch, R. A.; Robertson, J. E. Difluorodiazirine. V. Difluoromethyl esters and ethers. J. Heterocycl. Chem. 1965, 2, 152– 156, DOI: 10.1002/jhet.5570020209[Crossref], [CAS], Google Scholar113https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF2MXktlWntr4%253D&md5=1188bac34dd03f3018f85f4a45745af3Difluorodiazirine. V. Difluoromethyl esters and ethersMitsch, Ronald A.; Robertson, Jerry E.Journal of Heterocyclic Chemistry (1965), 2 (2), 152-6CODEN: JHTCAD; ISSN:0022-152X.cf. CA 62, 13055h. The preparation of difluoromethyl esters and ethers by the photolytic reaction of difluorodiazirine with acids and alcohols, respectively, is described. Difluoromethyl benzoate is converted, under mild conditions, to methyl benzoate, benzamide, and benzanilide by reaction with methanol, ammonium hydroxide, and aniline, respectively. The difluoromethyl esters arc activated to nucleophilic reaction by virtue of the α-fluorine substitution.114Neuberger, J.; Mieli-Vergani, G.; Tredger, J. M.; Davis, M.; Williams, R. Oxidative metabolism of halothane in the production of altered hepatocyte membrane antigens in acute halothane-induced hepatic necrosis. Gut 1981, 22, 669– 672, DOI: 10.1136/gut.22.8.669[Crossref], [PubMed], [CAS], Google Scholar114https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38Xmt1Giug%253D%253D&md5=44107db5a0287cd29c095fa2ad711e4aOxidative metabolism of halothane in the production of altered hepatocyte membrane antigens in acute halothane-induced hepatic necrosisNeuberger, J.; Mieli-Vergani, Giorgina; Tredger, J. M.; Davis, M.; Williams, RogerGut (1981), 22 (8), 669-72CODEN: GUTTAK; ISSN:0017-5749.The expression of antigens on halothane [151-67-7]-altered hepatocytes was assocd. with the oxidative metab. of halothane. The susceptibility of halothane-altered cells to cytotoxicity by normal lymphocytes decreased with decreasing O tension at anesthesia. This result contrasts with those of other groups which showed that the reductive route was involved in the direct hepatotoxic reaction attributed to halothane.115McLain, G. E.; Sipes, I. G.; Brown, B. R., Jr. An animal model of halothane hepatotoxicity: roles of enzyme induction and hypoxia. Anesthesiology 1979, 51, 321– 326, DOI: 10.1097/00000542-197910000-00008[Crossref], [PubMed], [CAS], Google Scholar115https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3cXkvFU%253D&md5=97a82c2d1e394dd07137fe025f6fed5fAn animal model of halothane hepatotoxicity: roles of enzyme induction and hypoxiaMcLain, George E.; Sipes, I. Glenn; Brown, Burnell R., Jr.Anesthesiology (1979), 51 (4), 321-6CODEN: ANESAV; ISSN:0003-3022.Exposure of phenobarbital-pretreated male rats to halothane [151-67-7] (1%, for 2 h under conditions of hypoxia) resulted in extensive centrilobular necrosis within 24 h. Accompanying the morphologic damage were an increase in serum glutamic-pyruvic transaminase [9000-86-6] and a decrease in the hepatic microsomal cytochrome P-450 [9035-51-2]. Glutathione [70-18-8] levels in the liver were unchanged. Phenobarbital-pretreated rats anesthetized with halothane at higher O2 concns. had only minor morphologic changes at 24 h. Hepatic injury was not apparent in any nonphenobarbital-induced rat or in any induced animal exposed to ether [60-29-7] under hypoxic conditions. There was a 2.6-fold increase in the 24-h urinary excretion of F in those rats in which extensive centrilobular necrosis developed. The in vivo covalent binding to lipids of 14C from halothane-14C also was increased markedly when halothane-14C was administered i.p. to phenobarbital-induced rats maintained hypoxic for 2 h. Thus halothane is metabolized to hepatotoxic intermediates by a reductive or nonoxygen-dependent cytochrome P-450-dependent pathway. This animal model of halothane-induced hepatotoxicity may be clin. relevant. A decrease in hepatic blood flow during halothane anesthesia may decrease the PO2 available to hepatocytes and thus direct the metab. of halothane along its reductive, hepatotoxic pathway.116Ray, D. C.; Drummond, G. B. Halothane hepatitis. Br. J. Anaesth. 1991, 67, 84– 99, DOI: 10.1093/bja/67.1.84[Crossref], [PubMed], [CAS], Google Scholar116https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXmt1amsrg%253D&md5=b194e93271ea0709c6ed14cb1455b9c6Halothane hepatitisRay, D. C.; Drummond, G. B.British Journal of Anaesthesia (1991), 67 (1), 84-99CODEN: BJANAD; ISSN:0007-0912.A review with 187 refs.117Bourdi, M.; Chen, W.; Peter, R. M.; Martin, J. L.; Buters, J. T.; Nelson, S. D.; Pohl, L. R. Human cytochrome P450 2E1 is a major autoantigen associated with halothane hepatitis. Chem. Res. Toxicol. 1996, 9, 1159– 1166, DOI: 10.1021/tx960083q[ACS Full Text], [CAS], Google Scholar117https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XlslOksrg%253D&md5=f7e3cba9321de99ed160db4f31d00e27Human Cytochrome P450 2E1 Is a Major Autoantigen Associated with Halothane HepatitisBourdi, Mohammed; Chen, Weiqiao; Peter, Raimund M.; Martin, Jackie L.; Buters, Jeroen T. M.; Nelson, Sidney D.; Pohl, Lance R.Chemical Research in Toxicology (1996), 9 (7), 1159-1166CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)Autoantibodies against specific human cytochrome P450s have been found in the sera of patients suffering from a variety of diseases, including those caused by drugs. In the cases of tienilic acid- and dihydralazine-induced hepatitis, patients have serum autoantibodies directed against cytochromes P 450 2C9 and P 450 1A2, resp. In the present study, we have found that 25 of 56 (45%) patients diagnosed with halothane hepatitis have autoantibodies that react with human cytochrome P 450 2E1 that was purified from a baculovirus expression system. The autoantibodies inhibited the activity of cytochrome P 450 2E1 and appeared to be directed against mainly conformational epitopes. In addn., because cytochrome P 450 2E1 became trifluoroacetylated when it oxidatively metabolized halothane, it is possible that the covalently altered form of cytochrome P 450 2E1 may be able to bypass the immunol. tolerance that normally exists against cytochrome P 450 2E1. A similar mechanism may explain the formation of autoantibodies that have been found against other cellular targets of the reactive trifluoroacetyl chloride metabolite of halothane.118Kurth, M. J.; Yokoi, T.; Gershwin, M. E. Halothane-induced hepatitis: paradigm or paradox for drug-induced liver injury. Hepatology 2014, 60, 1473– 1475, DOI: 10.1002/hep.27253[Crossref], [PubMed], [CAS], Google Scholar118https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvVansbfI&md5=4d713af06e9f8ee9972a02b0d799f5dcHalothane-induced hepatitis: Paradigm or paradox for drug-induced liver injuryKurth, Mark J.; Yokoi, Tsuyoshi; Gershwin, M. EricHepatology (Hoboken, NJ, United States) (2014), 60 (5), 1473-1475CODEN: HPTLD9; ISSN:0270-9139. (John Wiley & Sons, Inc.)A review. This article describes about cellular events and signaling pathways underlying halothane-induced liver injury with emphasis on role of thymic stromal lymphopoietin receptor and cytokines such as interleukin-4 in pathogenesis in human and mouse.119Nastainczyk, W.; Ullrich, V.; Sies, H. Effect of oxygen concentration on the reaction of halothane with cytochrome P450 in liver microsomes and isolated perfused rat liver. Biochem. Pharmacol. 1978, 27, 387– 392, DOI: 10.1016/0006-2952(78)90366-0[Crossref], [PubMed], [CAS], Google Scholar119https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXkslSlt7o%253D&md5=adb0b275ee78f7481aa13072f2849e66Effect of oxygen concentration on the reaction of halothane with cytochrome P450 in liver microsomes and isolated perfused rat liverNastainczyk, Wolfgang; Ullrich, Volker; Sies, HelmutBiochemical Pharmacology (1978), 27 (4), 387-92CODEN: BCPCA6; ISSN:0006-2952.Formation of the complex between reduced cytochrome P450 [9035-51-2] and trifluoromethyl carbene [2441-28-3], formed in hepatic microsomal halothane [151-67-7] metab., was inhibited at high O concns. but began at <50μM O and was max. under anaerobic conditions. Metyrapone was an efficient inhibitor of the carbene complex formation. Organ spectrophotometry of isolated perfused livers showed that the complex appeared under slightly hypoxic conditions and that addn. of metyrapone abolished its formation. Halothane may be metabolized in vivo to reactive intermediates when the O concn. of the cell is <50μM.120Ahr, H. J.; King, L. J.; Nastainczyk, W.; Ullrich, V. The mechanism of reductive dehalogenation of halothane by liver cytochrome P450. Biochem. Pharmacol. 1982, 31, 383– 390, DOI: 10.1016/0006-2952(82)90186-1[Crossref], [PubMed], [CAS], Google Scholar120https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XktVSitbY%253D&md5=2b7cca80a26a1f6737878863cd0184aaThe mechanism of reductive dehalogenation of halothane by liver cytochrome P-450Ahr, Hans J.; King, L. J.; Nastainczyk, W.; Ullrich, V.Biochemical Pharmacology (1982), 31 (3), 383-90CODEN: BCPCA6; ISSN:0006-2952.Studies of the reductive dehalogenation of halothane (I) [151-67-7] leading to 2-chloro-1,1,1-trifluoroethane (II) [75-88-7] and 2-chloro-1,1-difluoroethylene (III) [359-10-4] in vitro in anaerobic rat liver microsomes showed that stimulation of NADPH oxidn. by I as well as formation of II and III were dependent on cytochrome P-450 (IV) [9035-51-2]. After replacement of NADPH by Na dithionite as a reducing agent, III was the only product. The product pattern was influenced by 3-methylcholanthrene, benzo[a]pyrene, phenobarbitone, and Arochlor 1254 pretreatments and by addn. of anti-IV-PB Ig. II/III ratio was shifted by addn. or inhibition of cytochrome b5 and by pH variation, indicating a crucial role of the second electron donation to IV in detg. product pattern. The intermediate complex of IV with a Soret band at 470 nm formed with I in reduced liver microsomes decompd. spontaneously to give III; the 470-nm peak may represent IV Fe3+--CHCl-CF3 carbanion complex. A reaction path was derived including radical and carbanion intermediates as reactive precursors of II and III, resp.121Baker, M. T.; Van Dyke, R. A. Reductive halothane metabolite formation and halothane binding in rat hepatic microsomes. Chem.-Biol. Interact. 1984, 49, 121– 132, DOI: 10.1016/0009-2797(84)90056-5[Crossref], [PubMed], [CAS], Google Scholar121https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXktlaku7w%253D&md5=3dd71e3ba602de20e9b1b9134c68c778Reductive halothane metabolite formation and halothane binding in rat hepatic microsomesBaker, Max T.; Van Dyke, Russell A.Chemico-Biological Interactions (1984), 49 (1-2), 121-32CODEN: CBINA8; ISSN:0009-2797.The formation of the reductive 14C-labeled halothane [151-67-7] metabolites, 2-chloro-1,1,1-trifluoroethane (CTE) [75-88-7] and 2-chloro-1,1-difluoroethylene (CDE) [359-10-4], was detd. in anaerobic microsomal incubations by HPLC. The HPLC technique used allowed accurate measurements of low levels of [14C]halothane metabolites. Comparisons of metabolic profiles and halothane binding in microsomes reduced with NADPH and Na dithionite showed that dithionite stimulated CDE prodn. and total halothane degrdn., but inhibited CTE formation and [14C]-halothane binding. Similarly, the addn. of isoflurane [26675-46-7], but not enflurane [13838-16-9], to microsomes increased CDE prodn. and decreased CTE formation and [14C]halothane-lipid binding. Measurement of F- in similar incubations show that F- release from halothane correlates with the formation of CDE and not CTE. The relative prodn. of CTE and CDE may not remain const. in microsomal prepns., and halothane binding correlates with CTE formation and not CDE and F- prodn.122Van Dyke, R. A.; Baker, M. T.; Jansson, I.; Schenkman, J. Reductive metabolism of halothane by purified cytochrome P-450. Biochem. Pharmacol. 1988, 37, 2357– 2361, DOI: 10.1016/0006-2952(88)90361-9[Crossref], [PubMed], [CAS], Google Scholar122https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXksVOrs7Y%253D&md5=f051cdde40286e63f9ad7f997a6cc5c9Reductive metabolism of halothane by purified cytochrome P-450Van Dyke, Russell A.; Baker, Max T.; Jansson, Ingela; Schenkman, JohnBiochemical Pharmacology (1988), 37 (12), 2357-61CODEN: BCPCA6; ISSN:0006-2952.The reductive metab. of halothane was detd. by using purified RLM2, PBRLM4 and PBRLM5 forms of rat liver microsomal cytochrome P 450. The metabolites, 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE), were detd. All 3 forms of cytochrome P 450 produced CTE with relatively small differences in its prodn. among the various forms. There were major differences, however, in the prodn. of CDE, with PBRLM5 being the most active. PBRLM5 was also the only form to show the development of a complex between halothane and cytochrome P 450. This complex absorbed light maximally at 470 nm. The complex formation and the prodn. of CDE by PBRLM5 were stimulated by the addn. of cytochrome b5. Cytochrome b5 had no effect on CDE prodn. by PBRLM4 and inhibited the prodn. of both CTE and CDE by RLM2. The 2-electron redn. of halothane by cytochrome P 450 was catalyzed by the PBRLM5 form and cytochrome b5 stimulated the transfer of the 2nd electron to halothane through PBRLM5, but not RLM2 or PBRLM4.123Trudell, J. R.; Bosterling; Trevor, A. 1-Chloro-2,2,2-trifluoroethyl radical: Formation from halothane by human cytochrome P-450 in reconstituted vesicles and binding to phospholipids. Biochem. Biophys. Res. Commun. 1981, 102, 372– 377, DOI: 10.1016/0006-291X(81)91531-X[Crossref], [PubMed], [CAS], Google Scholar123https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXlvF2nsbg%253D&md5=41f098c996edd86bf03c48996390dbe81-Chloro-2,2,2-trifluoroethyl radical: formation from halothane by human cytochrome P-450 in reconstituted vesicles and binding to phospholipidsTrudell, James R.; Boesterling, Bernhard; Trevor, AnthonyBiochemical and Biophysical Research Communications (1981), 102 (1), 372-7CODEN: BBRCA9; ISSN:0006-291X.The first complete structural characterization of a metabolically-produced halocarbon radical bound to a phospholipid is reported. Human cytochrome P-450 and NADPH cytochrome P-450 reductase were reconstituted into vesicles composed of dioleoylphosphatidylcholine and egg phosphatidylethanolamine. The vesicles were incubated under Ar with NADPH and [14C]halothane (1-[14C]-2-bromo-2-chloro-1,1,1-trifluoroethane), and the dioleoylphosphatidylcholine fraction was isolated and subjected to transesterification. Sepn. of the resulting fatty acid Me esters resulted in 1 radioactive fraction which gas chromatog.-mass spectrometry revealed to be a mixt. of 9- and 10-(1-chloro-2,2,2-trifluoroethyl)-stearate Me ester.124Pohl, L. R.; Kenna, J. G.; Satoh, H.; Christ, D.; Martin, J. L. Neoantigens associated with halothane hepatitis. Drug Metab. Rev. 1989, 20, 203– 217, DOI: 10.3109/03602538909103537[Crossref], [PubMed], [CAS], Google Scholar124https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXhvQ%253D%253D&md5=eae09f2a981df2b150bcadca6c7de3dfNeoantigens associated with halothane hepatitisPohl, Lance R.; Kenna, J. G.; Satoh, Hiroko; Christ, David; Martin, Jackie L.Drug Metabolism Reviews (1989), 20 (2-4), 203-17CODEN: DMTRAR; ISSN:0360-2532.A review with 33 refs. with discussion on the current status of the nature of the neoantigens assocd. with halothane hepatitis. It is believed that the approaches used to characterize these neoantigens can be applied to the study of the neoantigens assocd. with other drug toxicities believed to have an immunopathol. etiol.125Brown, A. P.; Hastings, K. L.; Gandolfi, A. J.; Brendel, K. Covalent binding of a halothane metabolite and neoantigen production in guinea pig liver slices. Adv. Exp. Med. Biol. 1991, 283, 693– 697, DOI: 10.1007/978-1-4684-5877-0_89[Crossref], [PubMed], [CAS], Google Scholar125https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXlslCnt7c%253D&md5=390bf5d89a3a1ae19487724bb9b33abbCovalent binding of a halothane metabolite and neoantigen production in guinea pig liver slicesBrown, Alan P.; Hastings, Kenneth L.; Gandolfi, A. Jay; Brendel, KlausAdvances in Experimental Medicine and Biology (1991), 283 (Biol. React. Intermed. 4), 693-7CODEN: AEMBAP; ISSN:0065-2598.Halothane hepatitis is a rare and potentially fatal consequence to the use of this anesthetic. The physiol. basis of the disease appears to be an immune response to neoantigens formed by the covalent binding of halothane metabolites to liver protein. Liver slices were used to study the condition for halothane assocd. neoantigen formation in vitro. Liver slices, (1 cm diam., 300 μm thick) from male Harley guinea pigs (600 g) were exposed to either 1.0 or 1.7 mM halothane (media Harley guinea pigs (600 g) were exposed to either 1.0 or 1.7 mM halothane (media concn.) in 95% O2/5% CO2 for 12 h. Covalent binding was detd. using 14C-halothane. Neoantigens were detected by western immunoblot assay using rabbit anti-trifluoroacetylated albumin antiserum. Covalent binding was detected by 1 h of incubation and increased linearly through 12 h. Covalent binding preceded and correlated with the appearance of neoantigen. By 12 h of incubation, 5 neoantigens were seen with mol. wts. ranging from 51-97 kD. These neoantigens have mol. wts. similar to those seen in vivo. This in vitro model system can be used to examine the mechanism for covalent binding and neoantigen prodn. in the hepatocyte.126Brown, A. P.; Hastings, K. L.; Gandolfi, A. J.; Liebler, D. C.; Brendel, K. Formation and identification of protein adducts to cytosolic proteins in guinea pig liver slices exposed to halothane. Toxicology 1992, 73, 281– 295, DOI: 10.1016/0300-483X(92)90070-U[Crossref], [PubMed], [CAS], Google Scholar126https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xlt1Kisrc%253D&md5=3043e5ae3c5da824fcb679d75ead9d04Formation and identification of protein adducts to cytosolic proteins in guinea pig liver slices exposed to halothaneBrown, Alan P.; Hastings, Kenneth L.; Gandolfi, A. Jay; Liebler, Daniel C.; Brendel, KlausToxicology (1992), 73 (3), 281-95CODEN: TXCYAC; ISSN:0300-483X.An in vitro liver slice system was used to study the formation of protein adducts following exposure to halothane. Liver slices (30-35 mg wet wt., 250-300 μm thick) from adult male Hartley guinea pigs (600-800 g) were exposed to [14C]halothane (0.6-0.9 μCi, 1.0-1.7 mM) in 95% O2/5% CO2 for 1, 6 and 12 h. The slices were homogenized and subcellular fractions prepd. Proteins were resolved by electrophoresis and bound radioactivity was detected by scintillation counting and autoradiog. Greater than 80% of detectable radioactivity to whole liver cell protein was localized in the 20-30 kDa range and increased in a linear fashion over the 12-h incubation period. Covalent binding was localized to two proteins of 27 kDa and 26 kDa present in the cytosolic compartment. Purifn. followed by N-terminal amino acid sequence anal. of the 27-kDa protein has identified it to be homologous with glutathione S-transferase. This cytosolic protein appears to be the major target for trifluoroacetylation in liver slices exposed to halothane.127Harris, J. W.; Pohl, L. R.; Martin, J. L.; Anders, M. W. Tissue acylation by the chlorofluorocarbon substitute 2,2-dichloro-1,1,1-trifluoroethane. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 1407– 1410, DOI: 10.1073/pnas.88.4.1407[Crossref], [PubMed], [CAS], Google Scholar127https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXhtFKmu7Y%253D&md5=a0503049d6cb83b8bdca918b54b78988Tissue acylation by the chlorofluorocarbon substitute 2,2-dichloro-1,1,1-trifluoroethaneHarris, James W.; Pohl, Lance R.; Martin, Jackie L.; Anders, M. W.Proceedings of the National Academy of Sciences of the United States of America (1991), 88 (4), 1407-10CODEN: PNASA6; ISSN:0027-8424.Here the authors report studies on the bioactivation of the chlorofluorocarbon substitute 2,2-dichloro-1,1,1-trifluoroethane (HCFC-123) to an electrophilic intermediate that reacts covalently with liver proteins. HCFC-123 and its analog halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) were studied in rats by 19F NMR spectroscopy, and it was found that a trifluoroacetylated lysine adduct was formed with liver proteins. Also, the pattern of proteins immunoreactive with hapten-specific antitrifluoroacetylprotein antibodies was identical in livers of HCFC-123- and halothane-exposed rats. Because halothane causes an idiosyncratic, and sometimes fatal, hepatitis that is assocd. with an immune response against several trifluoroacetylated liver proteins, the present findings raise the possibility that humans exposed to HCFC-123 or structurally related hydrochlorofluorocarbons may be at risk of developing an immunol. mediated hepatitis.128Pohl, L. R. An immunochemical approach of identifying and characterizing protein targets of toxic reactive metabolites. Chem. Res. Toxicol. 1993, 6, 786– 793, DOI: 10.1021/tx00036a006[ACS Full Text], [CAS], Google Scholar128https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXmsVejsLs%253D&md5=30dd61034178e27597f0202a2dee0f0eAn immunochemical approach of identifying and characterizing protein targets of toxic reactive metabolitesPohl, Lance R.Chemical Research in Toxicology (1993), 6 (6), 786-93CODEN: CRTOEC; ISSN:0893-228X.A review with 101 refs. on protein adducts assocd. with halothane hepatitis, other uses of anti-TFA antibodies, protein adducts assocd. with acetaminophen hepatotoxicity, protein adducts assocd. with ethanol hepatotoxicity, protein adducts assocd. with diclofenac hepatotoxicity, and proteins posttranslationally modified by products of oxidative stress.129Kenna, J. G.; Satoh, H.; Christ, D. D.; Pohl, L. R. Metabolic basis for a drug hypersensitivity: antibodies in sera from patients with halothane hepatitis recognize liver neoantigens that contain the trifluoroacetyl group derived from halothane. J. Pharmacol. Exp. Ther. 1988, 245, 1103– 1109[PubMed], [CAS], Google Scholar129https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXks1KktbY%253D&md5=23dad774921d26a4aaa46b06e7a1a748Metabolic basis for a drug hypersensitivity: antibodies in sera from patients with halothane hepatitis recognize liver neoantigens that contain the trifluoroacetyl group derived from halothaneKenna, J. G.; Satoh, H.; Christ, D. D.; Pohl, L. R.Journal of Pharmacology and Experimental Therapeutics (1988), 245 (3), 1103-9CODEN: JPETAB; ISSN:0022-3565.Liver microsomes form rats treated in vivo with halothane or deuterated halothane were tested by immunoblotting for reactivity with patients' sera and with an antiserum specific for the covalently bound trifluoroacetyl (TFA) halide metabolite of halothane. Rat liver microsomes incubated aerobically or anaerobically with halothane or deuterated halothane in vitro, ± NADPH and/or NADH, were also analyzed. Neoantigen expression involved oxidative halothane metab. by cytochromes P 450 to TFA halide and covalent binding of the TFA group to the proteins. Incubation of microsomes from halothane-treated rats with 1 M piperidine cleaved the TFA groups from the proteins and abolished antigenicity, confirming this conclusion. Recognition of the neoantigens by the patients' antibodies was inhibited only partially by the hapten deriv. N-E-TFA-Dl-lysine. It appears that the patients' antibodies recognize epitopes consisting of the TFA group plus assocd. structural features of the protein carriers (100 kilodalton (kDa), 76 kDa, 59 kDa, 57 kDa, and 54 kDa), not the TFA hapten alone. The approach described may be of general utility for characterization of drug-induced neoantigens assocd. with other drug hypersensitivities.130Kenna, J. G.; Neuberger, J.; Williams, R. Evidence for expression in human liver of halothane-induced neoantigens recognized by antibodies in sera from patients with halothane hepatitis. Hepatology 1988, 8, 1635– 1641, DOI: 10.1002/hep.1840080627[Crossref], [PubMed], [CAS], Google Scholar130https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaL1M%252FltlansA%253D%253D&md5=d41d0e21185e3bcd138268cb3fb20805Evidence for expression in human liver of halothane-induced neoantigens recognized by antibodies in sera from patients with halothane hepatitisKenna J G; Neuberger J; Williams RHepatology (Baltimore, Md.) (1988), 8 (6), 1635-41 ISSN:0270-9139.Previous investigations have shown that antibodies in sera from patients with halothane hepatitis recognize neoantigens, expressed in livers of halothane-exposed rabbits and rats, which consist of a halothane metabolite bound covalently to specific microsomal proteins. These studies have suggested that the patients' antibodies may play a role in the pathogenesis of the hepatitis. In the present investigation, human liver biopsy samples were analyzed using an immunoblotting method to seek evidence for expression of halothane-induced neoantigens in humans. Sera from four patients with halothane hepatitis, which recognized halothane-induced rabbit liver neoantigens of 100, 76 and 57 kD, reacted strongly with antigens of very similar molecular weights that were expressed in livers from two patients who had died of cardiac failure following recent anesthesia with halothane. The antigens were not expressed in normal human liver or in livers from three patients who died of cardiac failure following anesthesia with agents other than halothane. The human antigens were not recognized by antibodies present in various control sera. Recognition of the 100- and 76-kD human antigens by the patients' antibodies was greatly reduced by absorption of sera with liver microsomes from halothane-exposed rabbits, but not by absorption of sera with control rabbit microsomes. These results indicate that humans exposed to halothane express liver neoantigens which are analogous to the halothane metabolite-protein neoantigens characterized previously in halothane-exposed animals.131Behne, M.; Wilke, H. J.; Harder, S. Clinical pharmacokinetics of sevoflurane. Clin. Pharmacokinet. 1999, 36, 13– 26, DOI: 10.2165/00003088-199936010-00002[Crossref], [PubMed], [CAS], Google Scholar131https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXht1WgsbY%253D&md5=038daca584abfd368886530bd6a83d53Clinical pharmacokinetics of sevofluraneBehne, Michael; Wilke, Hans-Joachim; Harder, SebastianClinical Pharmacokinetics (1999), 36 (1), 13-26CODEN: CPKNDH; ISSN:0312-5963. (Adis International Ltd.)Sevoflurane is a comparatively recent addn. to the range of inhalational anesthetics which has been recently released for clin. use. In comparison to older inhalational agents such as isoflurane or halothane, the most important property of sevoflurane is its low soly. in the blood. This results in a more rapid uptake and induction than the "older" inhalational agents, improved control of depth of anesthesia and faster elimination and recovery. The more rapid pharmacokinetics are a result of the low blood/gas partition coeff. of 0.69. With an oil/gas partition coeff. of 47.2, the min. alveolar concn. (MAC) of sevoflurane is 2.05%. Two to 5% of the drug taken up is metabolized by the liver. The pharmacokinetics of sevoflurane do not change in children, obese patients or patients with renal insufficiency. The pharmacokinetics and pleasant odor of sevoflurane make mask induction feasible, which is an obvious advantage in pediatric anesthesia. The hepatic metab. of sevoflurane results in the formation of inorg. fluoride. Upon contact with alk. CO2 absorbent, a small amt. of sevoflurane is degraded and a metabolite (compd. A) is formed and inhaled in trace amts. Whether inorg. fluoride or compd. A are nephrotoxic is presently a matter of controversy.132Kharasch, E. D.; Karol, M. D.; Lanni, C.; Sawchuk, R. Clinical sevoflurane metabolism and disposition. I. Sevoflurane and metabolite pharmacokinetics. Anesthesiology 1995, 82, 1369– 1378, DOI: 10.1097/00000542-199506000-00008[Crossref], [PubMed], [CAS], Google Scholar132https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK2Mzhtl2lug%253D%253D&md5=1b52ac6489fe4d61e555aa26de0cfc6eClinical sevoflurane metabolism and disposition. I. Sevoflurane and metabolite pharmacokineticsKharasch E D; Karol M D; Lanni C; Sawchuk RAnesthesiology (1995), 82 (6), 1369-78 ISSN:0003-3022.BACKGROUND: Sevoflurane has low blood and tissue solubility and is metabolized to free fluoride and hexafluoroisopropanol (HFIP). Although sevoflurane uptake and distribution and fluoride formation have been described, the pharmacokinetics of HFIP formation and elimination are incompletely understood. This investigation comprehensively characterized the simultaneous disposition of sevoflurane, fluoride, and HFIP. METHODS: Ten patients within 30% of ideal body weight who provided institutional review board-approved informed consent received sevoflurane (2.7% end-tidal, 1.3 MAC) in oxygen for 3 h after propofol induction, after which anesthesia was maintained with propofol, fentanyl, and nitrous oxide. Sevoflurane and unconjugated and total HFIP concentrations in blood were determined during anesthesia and for 8 h thereafter. Plasma and urine fluoride and total HFIP concentrations were measured during and through 96 h after anesthetic administration. Fluoride and HFIP were quantitated using an ion-selective electrode and by gas chromatography, respectively. RESULTS: The total sevoflurane dose, calculated from the pulmonary uptake rate, was 88.8 +/- 9.1 mmol. Sevoflurane was rapidly metabolized to the primary metabolites fluoride and HFIP, which were eliminated in urine. HFIP circulated in blood primarily as a glucuronide conjugate, with unconjugated HFIP < or = 15% of total HFIP concentrations. In blood, peak unconjugated HFIP concentrations were less than 1% of peak sevoflurane concentrations. Apparent renal fluoride and HFIP clearances (mean +/- SE) were 51.8 +/- 4.5 and 52.6 +/- 6.1 ml/min, and apparent elimination half-lives were 21.4 +/- 2.8 and 20.1 +/- 2.6 h, respectively. Renal HFIP and net fluoride excretion were 4,300 +/- 540 and 3,300 +/- 540 mumol. Compared with the estimated sevoflurane uptake, 4.9 +/- 0.5% of the dose taken up was eliminated in the urine as HFIP. For fluoride, 3.7 +/- 0.4% of the sevoflurane dose taken up was eliminated in the urine, which, because a portion of fluoride is sequestered in bone, corresponded to approximately 5.6% of the sevoflurane dose metabolized to fluoride. CONCLUSIONS: Sevoflurane was rapidly metabolized to fluoride and HFIP, which was rapidly glucuronidated and eliminated in the urine. The overall extent of sevoflurane metabolism was approximately 5%.133Kharasch, E. D.; Armstrong, A. S.; Gunn, K.; Artru, A.; Cox, K.; Karol, M. D. Clinical sevoflurane metabolism and disposition. II. The role of cytochrome P450 2E1 in fluoride and hexafluoroisopropanol formation. Anesthesiology 1995, 82, 1379– 1388, DOI: 10.1097/00000542-199506000-00009[Crossref], [PubMed], [CAS], Google Scholar133https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK2Mzhtl2luw%253D%253D&md5=373c22f4ccb4ff24b6df206307dc67d1Clinical sevoflurane metabolism and disposition. II. The role of cytochrome P450 2E1 in fluoride and hexafluoroisopropanol formationKharasch E D; Armstrong A S; Gunn K; Artru A; Cox K; Karol M DAnesthesiology (1995), 82 (6), 1379-88 ISSN:0003-3022.BACKGROUND: Sevoflurane is metabolized to free fluoride and hexafluoroisopropanol (HFIP). Cytochrome P450 2E1 is the major isoform responsible for sevoflurane metabolism by human liver microsomes in vitro. This investigation tested the hypothesis that P450 2E1 is predominantly responsible for sevoflurane metabolism in vivo. Disulfiram, which is converted in vivo to a selective inhibitor of P450 2E1, was used as a metabolic probe for P450 2E1. METHODS: Twenty-one patients within 30% of ideal body weight, who provided institutional review board-approved informed consent and were randomized to receive disulfiram (500 mg oral, n = 11) or nothing (control, n = 10) the night before surgery, were evaluated. All patients received sevoflurane (2.7% end-tidal, 1.3 MAC) in oxygen for 3 h after propofol induction. Thereafter, sevoflurane was discontinued, and anesthesia was maintained with propofol, fentanyl, and nitrous oxide. Blood sevoflurane concentrations during anesthesia and for 8 h thereafter were measured by gas chromatography. Plasma and urine fluoride and total (unconjugated plus glucuronidated) HFIP concentrations were measured by an ion-selective electrode and by gas chromatography, respectively, during anesthesia and for 96 h postoperatively. RESULTS: Patient groups were similar with respect to age, weight, sex, case duration, and intraoperative blood loss. The total sevoflurane dose, measured by cumulative end-tidal sevoflurane concentrations (3.7 +/- 0.1 MAC-h; mean +/- SE), total pulmonary uptake, and blood sevoflurane concentrations, was similar in both groups. In control patients, plasma fluoride and HFIP concentrations were increased compared to baseline values intraoperatively and postoperatively for the first 48 and 60 h, respectively. Disulfiram treatment significantly diminished this increase. Plasma fluoride concentrations increased from 2.1 +/- 0.3 microM (baseline) to 36.2 +/- 3.9 microM (peak) in control patients, but only from 1.7 +/- 0.2 to 17.0 +/- 1.6 microM in disulfiram-treated patients (P < 0.05 compared with control patients). Peak plasma HFIP concentrations were 39.8 +/- 2.6 and 14.4 +/- 1.1 microM in control and disulfiram-treated patients (P < 0.05), respectively. Areas under the plasma fluoride- and HFIP-time curves also were diminished significantly to 22% and 20% of control patients, respectively, by disulfiram treatment. Urinary excretion of fluoride and HFIP was similarly significantly diminished in disulfiram-treated patients. Cumulative 96-h fluoride and HFIP excretion in disulfiram-treated patient was 1,080 +/- 210 and 960 +/- 240 mumol, respectively, compared to 3,950 +/- 560 and 4,300 +/- 540 mumol in control patients (P < 0.05). CONCLUSIONS: Disulfiram, an effective P450 2E1 inhibitor, substantially decreased fluoride ion and HFIP production during and after sevoflurane anesthesia. These results suggest that P450 2E1 is a predominant P450 isoform responsible for human sevoflurane metabolism in vivo.134Kharasch, E. D. Biotransformation of sevoflurane. Anesth. Analg. 1995, 81, 27S– 38S, DOI: 10.1097/00000539-199512001-00005[Crossref], [PubMed], [CAS], Google Scholar134https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XmvV2rtQ%253D%253D&md5=5ebb6765156a1c6cc2398213af8a4bfbBiotransformation of sevofluraneKharasch, Evan D.Anesthesia & Analgesia (Baltimore) (1995), 81 (6S), S27-S38CODEN: AACRAT; ISSN:0003-2999. (Williams & Wilkins)A review with 68 refs. Biotransformation of volatile anesthetics is of considerable interest because of known assocns. between metab. of certain anesthetics and specific organ toxicities. Advances in enzyme biochem. and mol. biol. have illuminated similarities and differences between animal and human drug metabolizing enzymes, permitting both appropriate extrapolation of animal data to humans and reconciliation of discrepancies between animal and human sevoflurane studies.135Thummel, K. E.; Kharasch, E. D.; Podoll, T.; Kunze, K. Human liver microsomal enflurane defluorination catalyzed by cytochrome P-450 2E1. Drug Metab. Dispos. 1993, 21, 350– 357[PubMed], [CAS], Google Scholar135https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXisVKrurY%253D&md5=1e44133d539b2b04e71d6845f0f438aeHuman liver microsomal enflurane defluorination catalyzed by cytochrome P-450 2E1Thummel, Kenneth E.; Kharasch, Evan D.; Podoll, Terry; Kunze, KentDrug Metabolism and Disposition (1993), 21 (2), 350-7CODEN: DMDSAI; ISSN:0090-9556.The volatile anesthetic agent enflurane undergoes oxidative metab. in human liver, yielding both inorg. and org. fluoride metabolites. Numerous studies in animals indicate that cytochrome P 450 2E1 is a major catalyst for the defluorination reaction. However, the P 450 enzyme catalyzing enflurane metab. in humans has not been identified. Therefore, expts. were conducted to det. whether hepatic P 450 2E1 is a catalyst for the reaction in humans, and whether other constitutive or inducible isoforms might also be involved. Purified human liver P 450 2E1, reconstituted with cytochrome b5 and P 450 reductase, catalyzed enflurane defluorination at a rate of 9.3 nmol F-/nmol P 450/30 min, in contrast to a mean liver microsomal rate of 2.0 nmol F-/nmol P 450/30 min. The microsomal rate of defluorination for individual human livers correlated significantly with the microsomal content of P 450 2E1 protein, the rate of p-nitrophenol hydroxylation, and the rate of chlorzoxazone 6-hydroxylation. In addn., specific anti-P 450 2E1 IgG, at a concn. of 10 mg IgG/nmol P 450, inhibited the microsomal reaction by 80%. Finally, a series of P 450 isoform-specific chem. inhibitors of oxidative metab. were screened for their ability to block human microsomal enflurane defluorination. Only di-Et dithiocarbamate, a mechanism-based inhibitor of P 450 2E1, inhibited the reaction; this occurred to a degree comparable to the effect of anti-P 450 2E1 antibody. These results demonstrate that P 450 2E1 is the predominant, if not only, enzyme catalyzing enflurane defluorination in human liver.136Kharasch, E. D.; Thummel, K. E.; Mautz, D.; Bosse, S. Clinical enflurane metabolism by cytochrome P450 2E1. Clin. Pharmacol. Ther. 1994, 55, 434– 440, DOI: 10.1038/clpt.1994.53[Crossref], [PubMed], [CAS], Google Scholar136https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK2c3hsFarsw%253D%253D&md5=75c256e631e235ca79b283322d545e32Clinical enflurane metabolism by cytochrome P450 2E1Kharasch E D; Thummel K E; Mautz D; Bosse SClinical pharmacology and therapeutics (1994), 55 (4), 434-40 ISSN:0009-9236.BACKGROUND: Fluorinated ether anesthetic hepatotoxicity and nephrotoxicity are mediated by cytochrome P450-catalyzed oxidative metabolism. Metabolism of the volatile anesthetic enflurane to inorganic fluoride ion by human liver microsomes in vitro is catalyzed predominantly by the cytochrome P450 isoform CYP2E1. This investigation tested the hypothesis that P450 2E1 is also the isoform responsible for human enflurane metabolism in vivo. Disulfiram, which is converted in vivo to a selective inhibitor of P450 2E1, was used as a metabolic probe for P450 2E1. METHODS: Twenty patients undergoing elective surgery were randomized to receive disulfiram (500 mg orally; n = 10) or nothing (control subjects; n = 10) the evening before surgery. All patients received a standard anesthetic of enflurane (2.2% end-tidal) in oxygen for 3 hours. Blood enflurane concentrations were measured by gas chromatography. Plasma and urine fluoride concentrations were quantitated by ion-selective electrode. RESULTS: Patient groups were similar with respect to age, weight, gender, duration of surgery, and blood loss. Total enflurane dose, measured by cumulative end-tidal enflurane concentrations (3.9 to 4.1 MAC-hr) and by blood enflurane concentrations, was similar in both groups. Plasma fluoride concentrations increased from 3.6 +/- 1.5 mumol/L (baseline) to 24.3 +/- 3.8 mumol/L (peak) in untreated patients (mean +/- SE). Disulfiram treatment completely abolished the rise in plasma fluoride concentration. Urine fluoride excretion was similarly significantly diminished in disulfiram-treated patients. Fluoride excretion in disulfiram-treated patients was 62 +/- 10 and 61 +/- 12 mumol on days 1 and 2, respectively, compared with 1090 +/- 180 and 1200 +/- 220 mumol in control subjects (p < 0.05 on each day). CONCLUSIONS: Disulfiram prevented fluoride ion production after enflurane anesthesia. These results suggest that P450 2E1 is the predominant P450 isoform responsible for human clinical enflurane metabolism in vivo.137Kharasch, E. D.; Hankins, D. C.; Cox, K. Clinical isoflurane metabolism by cytochrome P450 2E1. Anesthesiology 1999, 90, 766– 771, DOI: 10.1097/00000542-199903000-00019[Crossref], [PubMed], [CAS], Google Scholar137https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXhvFKhur4%253D&md5=4b4c448ff90ee438102a0aafdf520d67Clinical isoflurane metabolism by cytochrome P450 2E1Kharasch, Evan D.; Hankins, Douglas C.; Cox, KathyAnesthesiology (1999), 90 (3), 766-771CODEN: ANESAV; ISSN:0003-3022. (Lippincott Williams & Wilkins)Some evidence suggests that isoflurane metab. to trifluoroacetic acid and inorg. fluoride by human liver microsomes in vitro is catalyzed by cytochrome P 450 2E1 (CYP2E1). This investigation tested the hypothesis that P 450 2E1 predominantly catalyzes human isoflurane metab. in vivo. Disulfiram, which is converted in vivo to a selective inhibitor of P 450 2E1, was used as a metabolic probe for P 450 2E1. Twenty-two elective surgery patients who provided institutionally-approved written informed consent were randomized to receive disulfiram (500 mg orally, N = 12) or nothing (controls, N = 10) the evening before surgery. All patients received a std. isoflurane anesthetic (1.5% end-tidal in oxygen) for 8 h. Urine and plasma trifluoroacetic acid and fluoride concns. were quantitated in samples obtained for 4 days postoperatively. Patient groups were similar with respect to age, wt., gender, duration of surgery, blood loss, and delivered isoflurane dose, measured by cumulative end-tidal isoflurane concns. (9.7-10.2 MAC-hr). Postoperative urine excretion of trifluoroacetic acid (days 1-4) and fluoride (days 1-3) was significantly (P < 0.05) diminished in disulfiram-treated patients. Cumulative 0-96 h excretion of trifluoroacetic acid and fluoride in disulfiram-treated patients was 34 ± 72 and 270 ± 70 μmoles (mean ± SD), resp., compared to 440 ± 360 and 1500 ± 800 μmoles in controls (P < 0.05 for both). Disulfiram also abolished the rise in plasma metabolite concns. Disulfiram, a selective inhibitor of human hepatic P 450 2E1, prevented 80-90% of isoflurane metab. These results suggest that P 450 2E1 is the predominant P 450 isoform responsible for human clin. isoflurane metab. in vivo.138Strum, D. P.; Johnson, B. H.; Eger, E. I. 2nd. Stability of sevoflurane in soda lime. Anesthesiology 1987, 67, 779– 781, DOI: 10.1097/00000542-198711000-00024[Crossref], [PubMed], [CAS], Google Scholar138https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXhtFWisg%253D%253D&md5=8d8faef9112c85c70c188ee729f719d0Stability of sevoflurane in soda limeStrum, David P.; Johnson, Brynte H.; Eger, Edmond I., IIAnesthesiology (1987), 67 (5), 779-81CODEN: ANESAV; ISSN:0003-3022.Stability of a halogenated volatile anesthetic is important because of the potential toxicity assocd. with the breakdown products. A temp.-dependent degrdn. of sevoflurane [(F3C)2CHOCH2F] in soda lime was obsd. At 22°, soda lime degraded 6.5% of the sevoflurane per h. The rate increased by 1.6% per h per degree rise in temp., reaching 57.4% degrdn. per h at 54°. In contrast, isoflurane was not degraded by soda lime. Halothane did not degrade at 22° or 37°, but did degrade (2.2% per h) at 54°.139Morio, M.; Fujii, K.; Satoh, N.; Imai, M.; Kawakami, U.; Mizuno, T.; Kawai, Y.; Ogasawara, Y.; Tamura, T.; Negishi, A.; Kumagai, Y.; Kawai, Y. Reaction of sevoflurane and its degradation products with soda lime. Toxicity of the byproducts. Anesthesiology 1992, 77, 1155– 1164, DOI: 10.1097/00000542-199212000-00017[Crossref], [PubMed], [CAS], Google Scholar139https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXpsVWrug%253D%253D&md5=7108d6c9e328915a816593c6c5f14740Reaction of sevoflurane and its degradation products with soda lime. Toxicity of the byproductsMorio, Michio; Fujii, Kohyu; Satoh, Nobukatsu; Imai, Masahiro; Kawakami, Urao; Mizuno, Takahiro; Kawai, Yoichiro; Ogasawara, Yasumasa; Tamura, Takashi; et al.Anesthesiology (1992), 77 (6), 1155-64CODEN: ANESAV; ISSN:0003-3022.Sevoflurane previously has been reported to undergo extensive degrdn. in the presence of soda lime. To more completely characterize the extent and significance of this reaction, the authors studied degrdn. of sevoflurane with and without soda lime, as well as the toxicity and mutagenicity of the degrdn. products. Two degrdn. products detected were CF2 = C(CF3)OCH2F (compd. A) and CH3OCF2CH(CF3)OCH2F (compd. B). During circulation of 1%, 2%, and 3% sevoflurane in a closed anesthesia circuit for 8 h, peak concns. of compd. A were 13.3, 30.2, and 42.1 ppm at 2 h, resp. The concns. of compd. B did not exceed 2 ppm. The temp. of the soda lime was 43.3° at 1 h and increased gradually to 47.9° after 8 h. In closed flasks with soda lime, the magnitude of the decrease in sevoflurane concns. (3%) and of the increase in compd. A concns. was temp. dependent. The peak concns. of compd. A at 23°, 37°, and 54° were 32.8 at 2 h, 46.6 at 0.5 h, and 78.5 ppm at 0.5 h, resp. The LC50 (50% lethal concn.) of compd. A in Wistar rats was 1090 ppm in males and 1050 ppm in females exposed for 1 h. The LC50 was 420 ppm in males and 400 ppm in females exposed for 3 h. The chronic toxicity of compd. A in Wistar rats was studied by exposing rats 24 times, for 3 h each, to initial concns. of 30, 60, or 120 ppm in a ventilated chamber. At all concns., there were no apparent effects other than a loss of body wt. in females (120 ppm) on the final day. Compd. A did not induce mutation on the reverse (Ames) test at <2500 μg/dish (culture medium 2.7 mL) with activation by S-9 mixt., and below 1250 μg/dish (culture medium 2.7 mL) without activation, in four strains of S. typhimurium and in 1 strain of E. coli. Exposure of fibroblasts to 7500 ppm of compd. A for 1 h did not induce structural changes. In a study of acute toxicity of compd. B, there was no toxicity in Wistar rats after 3 h of exposure at 2400 ppm. The reverse (Ames) test for compd. B was neg. at 625-1250 μg/dish. The authors conclude that sevoflurane is extensively degraded in the presence of soda lime and heat and the LC50 in rats of one degrdn. product is >10-fold the peak concn. reached during an 8-h closed circuit anesthetic.140Jin, L.; Davis, M. R.; Kharasch, E. D.; Doss, G. A.; Baillie, T. A. Identification in rat bile of glutathione conjugates of fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether, a nephrotoxic degradate of the anesthetic agent sevoflurane. Chem. Res. Toxicol. 1996, 9, 555– 561, DOI: 10.1021/tx950162m[ACS Full Text], [CAS], Google Scholar140https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xpt1agsg%253D%253D&md5=c5c91c6633610cc7a80c031ee5726086Identification in rat bile of glutathione conjugates of fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether, a nephrotoxic degradate of the anesthetic agent sevofluraneJin, Lixia; Davis, Margaret R.; Kharasch, Evan D.; Doss, George A.; Baillie, Thomas A.Chemical Research in Toxicology (1996), 9 (2), 555-61CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)Recent studies have indicated that the nephrotoxicity of fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether ("Compd. A"), a breakdown product of the inhaled anesthetic sevoflurane, may be mediated by a reactive intermediate(s) generated via the cysteine conjugate β-lyase pathway. To gain a better understanding of glutathione (GSH)-dependent metab. of Compd. A, the present study was carried out with the primary goal of detecting and characterizing Compd. A-GSH conjugates. By ionspray LC-MS/MS and NMR spectroscopy, a total of four GSH conjugates ("A1-A4") were identified from the bile of rats dosed i.p. with Compd. A. A1 and A2 were identified as two diastereomers of S-[1,1-difluoro-2-(fluoromethoxy)-2-(trifluoromethyl)ethyl]glutathione, while A3 and A4 were identified as (E)- and (Z)-S-[1-fluoro-2-(fluoromethoxy)-2-(trifluoromethyl)vinyl]glutathione, resp. Quant. analyses indicated that approx. 29% of the administered dose of Compd. A was excreted into the bile in the form of the above GSH conjugates over a period of 6 h. Studies conducted in vitro demonstrated that the reaction of Compd. A with GSH was catalyzed by both rat liver cytosolic and microsomal glutathione S-transferases (GST), with the two enzyme systems exhibiting different product selectivities. Formation of these GSH conjugates also occurred nonenzymically at an appreciable rate. These results indicate that spontaneous and enzyme-mediated conjugation with GSH represents a major pathway of metab. of Compd. A in rats. Conjugation of Compd. A with GSH in vivo appeared to be catalyzed preferentially by microsomal rather than cytosolic GST, based on comparison of biliary, microsomal, and cytosolic metabolic profiles. By analogy with other haloalkenes, further metab. of the corresponding cysteine conjugates of Compd. A by renal cysteine conjugate β-lyase may lead to the formation of reactive acylating agents, which would be expected to bind covalently to cellular macromols. and cause organ-selective nephrotoxicity.141Iyer, R. A.; Anders, M. W. Cysteine conjugate β-lyase-dependent biotransformation of the cysteine S-conjugates of the sevoflurane degradation product 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A). Chem. Res. Toxicol. 1997, 10, 811– 819, DOI: 10.1021/tx960196+[ACS Full Text], [CAS], Google Scholar141https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXktVSisrk%253D&md5=df9198d23d3512e8831bceb07db0b391Cysteine Conjugate β-Lyase-Dependent Biotransformation of the Cysteine S-Conjugates of the Sevoflurane Degradation Product 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (Compound A)Iyer, Ramaswamy A.; Anders, M. W.Chemical Research in Toxicology (1997), 10 (7), 811-819CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (I, Compd. A) is a fluoroalkene formed by the base-catalyzed degrdn. of sevoflurane and is nephrotoxic in rats. Fluoroalkene I is a structural analog of other nephrotoxic haloalkenes that undergo glutathione S-conjugate formation and cysteine S-conjugate β-lyase-dependent bioactivation to reactive intermediates. The present expts. were designed to study the β-lyase-dependent biotransformation of S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine (II) and S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine by 19F NMR and UV spectroscopy and GC/MS. Incubation of cysteine S-conjugate II with rat kidney cytosol or a pyridoxal model system showed the formation of inorg. fluoride, pyruvate, and 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid (III), the expected products of a β-lyase-catalyzed reaction. The ratio of fluoride to pyruvate ranged from 2.3 to 2.5. The amt. of acid III formed in the rat kidney cytosol and the pyridoxal model system was, however, less than 5% of the amt. of pyruvate formed. Incubation of conjugate II with rat kidney cytosol and anal. by 19F NMR spectroscopy showed resonances that were assigned to 3,3,3-trifluorolactic acid (IV); the formation of acid IV was obsd. in the pyridoxal model only after prolonged incubation (>18 h). Lactic acid IV was identified as a degrdn. product of acid 9. Cysteine S-conjugate III was not stable in pH 7.4 buffer and underwent a rapid cyclization reaction (t1/2 ≈ III min) to form 2-[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3-thiazole-4-carboxylic acid. These data show that fluoroalkene I-derived cysteine S-conjugates are substrates for renal β-lyase and that acid III is formed as a terminal product. Acid III is, however, unstable and affords lactic acid IV as a degrdn. product.142Iyer, R. A.; Frink, E. J., Jr.; Ebert, T. J.; Anders, M. W. Cysteine conjugate β-lyase-dependent metabolism of compound A (2-[fluoromethoxy]-1,1,3,3,3-pentafluoro-1-propene) in human subjects anesthetized with sevoflurane and in rats given compound A. Anesthesiology 1998, 88, 611– 618, DOI: 10.1097/00000542-199803000-00009[Crossref], [PubMed], [CAS], Google Scholar142https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXitFamtrs%253D&md5=386ee386aeeb5d0a53dcc54ade4727dfCysteine conjugate β-lyase-dependent metabolism of compound a (2-[fluoromethoxy]-1,1,3,3,3-pentafluoro-1-propene) in human subjects anesthetized with sevoflurane and in rats given compound AIyer, Ramaswamy A.; Frink, Edward J., Jr.; Ebert, Thomas J.; Anders, M. W.Anesthesiology (1998), 88 (3), 611-618CODEN: ANESAV; ISSN:0003-3022. (Lippincott-Raven Publishers)Sevoflurane undergoes Baralyme- or soda lime-catalyzed degrdn. in the anesthesia circuit to yield compd. A (2-[fluoromethoxy]-1,1,3,3,3-pentafluoro-1-propene), which is nephrotoxic in rats and undergoes metab. via the cysteine conjugate β-lyase pathway in those animals. The objective of these expts. was to test the hypothesis that compd. A undergoes β-lyase-dependent metab. in humans. Human volunteers were anesthetized with sevoflurane (1.25 min. alveolar concn., 3%, 2 l/min, 8 h) and thereby exposed to compd. A. Urine was collected at 24-h intervals for 72 h after anesthesia. Rats, which served as a pos. control, were given compd. A i.p., and urine was collected for 24 h afterward. Human and rat urine samples were analyzed by 19F NMR spectroscopy and gas chromatog.-mass spectrometry for the presence of compd. A metabolites. Anal. of human and rat urine showed the presence of the compd. A metabolites S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-NK-acetyl-L-cysteine, (E)- and (Z)-S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid, 3,3,3-trifluorolactic acid, and inorg. fluoride. The presence of 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid and 3,3,3-trifluorolactic acid in human urine was confirmed by gas chromatog.-mass spectrometry. The formation of compd. A-derived mercapturates shows that compd. A undergoes glutathione S-conjugate formation. The identification of 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid and 3,3,3-trifluorolactic acid in the urine of humans anesthetized with sevoflurane shows that compd. A undergoes β-lyase-dependent metab. Metabolite formation was qual. similar in both human volunteers anesthetized with sevoflurane, and thereby exposed to compd. A, and in rats given compd. A, indicating that compd. A is metabolized by the β-lyase pathway in both species.143Martin, J. L.; Laster, M. J.; Kandel, L.; Kerschmann, R. L.; Reed, G. F.; Eger, E. I., II. Metabolism of compound A by renal cysteine-S-conjugate β-lyase is not the mechanism of compound A-induced renal injury in the rat. Anesth. Analg. 1996, 82, 770– 774, DOI: 10.1213/00000539-199604000-00017[Crossref], [PubMed], [CAS], Google Scholar143https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xis1Wltrw%253D&md5=614ccdeec985860132be925d7d197fcbMetabolism of compound A by renal cysteine-S-conjugate β-lyase is not the mechanism of compound A-induced renal injury in the ratMartin, J. L.; Laster, M. J.; Kandel, L.; Kerschmann, R. L.; Reed, G. F.; Eger, E. I. IIAnesthesia & Analgesia (Baltimore) (1996), 82 (4), 770-4CODEN: AACRAT; ISSN:0003-2999. (Williams & Wilkins)Compd. A [CF2:C(CF3)OCH2F], a vinyl ether produced by CO2 absorbents acting on sevoflurane, can produce corticomedullary junction necrosis (injury to the outer stripe of the outer medullary layer, i.e., corticomedullary junction) in rats. Several halogenated alkenes produce a histol. similar corticomedullary necrosis by converting glutathione conjugates of these alkenes to halothionoacetyl halides. To test whether this mechanism explained the nephrotoxicity of Compd. A, we blocked three metabolic steps which would lead to formation of a halothionoacetyl halide: (1) we depleted glutathione by administering dl-buthionine-S,R-sulfoximine (BSO); (2) we blocked cysteine S-conjugate formation by administering acivicin (AT-125); and (3) we inhibited subsequent metab. by renal cysteine conjugate β-lyase to the nephrotoxic halothionoacetyl halides by administering aminooxyacetic acid (AOAA). These treatments were given alone or in combination to sep. groups of 10 or 20 Wistar rats before their exposure to Compd. A. We hypothesized that blocking these metabolic steps should decrease the injury produced by breathing 150 ppm of Compd. A for 3 h. However, we found either no change or an increase in renal injury, suggesting that this pathway mediates detoxification rather than toxicity. Our findings suggest that the cysteine-S-conjugate-mediated pathway is not the mechanism of Compd. A nephrotoxicity and, therefore, obsd. interspecies differences in the activity of this activating pathway may not be relevant in the prediction of the nephrotoxic potential of Compd. A in clin. practice.144Kharasch, E. D.; Hoffman, G. M.; Thorning, D.; Hankins, D. C.; Kilty, C. G. Role of the renal cysteine conjugate β-lyase pathway in inhaled compound A nephrotoxicity in rats. Anesthesiology 1998, 88, 1624– 1633, DOI: 10.1097/00000542-199806000-00027[Crossref], [PubMed], [CAS], Google Scholar144https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXkt1egsr8%253D&md5=cdd5d6e73c285ad91b3a06fd6d7cd21eRole of the renal cysteine conjugate β-lyase pathway in inhaled compound A nephrotoxicity in ratsKharasch, Evan D.; Hoffman, Gary M.; Thorning, David; Hankins, Douglas C.; Kilty, Cormac G.Anesthesiology (1998), 88 (6), 1624-1633CODEN: ANESAV; ISSN:0003-3022. (Lippincott-Raven Publishers)The sevoflurane degrdn. product compd. A is nephrotoxic in rats and undergoes metab. to glutathione and cysteine S-conjugates, with further metab. by renal cysteine conjugate β-lyase to reactive intermediates. Evidence suggests that toxicity is mediated by renal uptake of compd. A S-conjugates and metab. by β-lyase. Previously, inhibitors of the β-lyase pathway (aminooxyacetic acid and probenecid) diminished the nephrotoxicity of i.p. compd. A. This investigation detd. inhibitor effects on the toxicity of inhaled compd. A. Fischer 344 rats underwent 3 h of nose-only exposure to compd. A (0-220 ppm in initial dose-response expts. and 100-109 ppm in subsequent inhibitor expts.). The inhibitors (and targets) were probenecid (renal org. anion transport mediating S-conjugate uptake), acivicin (γ-glutamyl transferase), aminooxyacetic acid (renal β-lyase), and aminobenzotriazole (cytochrome P 450). Urine was collected for 24 h, and the animals were killed. Nephrotoxicity was assessed by histol. and biochem. markers (serum BUN and creatinine; urine vol.; and excretion of protein, glucose, and α-glutathione-S-transferase, a predominantly proximal tubular cell protein). Compd. A caused dose-related proximal tubular cell necrosis, diuresis, proteinuria, glucosuria, and increased α-glutathione-S-transferase excretion. The threshold for toxicity was 98-109 ppm (294-327 ppm-h). Probenecid diminished compd. A-induced glucosuria and excretion of α-glutathione-S-transferase and completely prevented necrosis. Aminooxyacetic acid diminished compd. A-dependent proteinuria and glucosuria but did not decrease necrosis. Acivicin increased nephrotoxicity of compd. A, and aminobenzotriazole had no consistent effect on nephrotoxicity of compd. A. Nephrotoxicity of inhaled compd. A in rats was assocd. with renal uptake of compd. A S-conjugates and cysteine conjugates metab. by renal β-lyase. Manipulation of the β-lyase pathway elicited similar results, whether compd. A was administered by inhalation or i.p. injection. Route of administration does not apparently influence nephrotoxicity of compd. A in rats.145Alauddin, M. M. Positron emission tomography (PET) imaging with 18F-based radiotracers. Am. J. Nucl. Med. Mol. Imaging 2012, 2, 55– 76[PubMed], [CAS], Google Scholar145https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtFOrtb4%253D&md5=55308a81f4916c4a9bf09d92b767c9e6Positron emission tomography (PET) imaging with 18F-based radiotracersAlauddin, Mian M.American Journal of Nuclear Medicine and Molecular Imaging (2012), 2 (1), 55-76CODEN: AJNMAU; ISSN:2160-8407. (e-Century Publishing Corp.)A review. Positron emission tomog. (PET) is a nuclear medicine imaging technique that is widely used in early detection and treatment follow up of many diseases, including cancer. This modality requires positron-emitting isotope labeled biomols., which are synthesized prior to perform imaging studies. Fluorine-18 is one of the several isotopes of fluorine that is routinely used in radiolabeling of biomols. for PET; because of its positron emitting property and favorable half-life of 109.8 min. The biol. active mol. most commonly used for PET is 2-deoxy-2-18F-fluoro-β--glucose (18F-FDG), an analog of glucose, for early detection of tumors. The concns. of tracer accumulation (PET image) demonstrate the metabolic activity of tissues in terms of regional glucose metab. and accumulation. Other tracers are also used in PET to image the tissue concn. In this review, information on fluorination and radiofluorination reactions, radiofluorinating agents, and radiolabeling of various compds. and their application in PET imaging is presented.146Pauwels, E. K. J. 18F-labeled fluorodeoxyglucose for PET imaging: the working mechanism and its clinical implication. Drugs Future 2001, 26, 659– 668, DOI: 10.1358/dof.2001.026.07.858710[Crossref], [CAS], Google Scholar146https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXot1CntL0%253D&md5=f75a1c778a0d193615cedb576573e82a18F-labeled fluorodeoxyglucose for PET imaging: The working mechanism and its clinical implicationPauwels, Ernest K. J.Drugs of the Future (2001), 26 (7), 659-668CODEN: DRFUD4; ISSN:0377-8282. (Prous Science)A review on the uptake mechanism and biochem. processing of Glc and fluorodeoxyglucose (FDG) in normal and malignant cells. Uptake of Glc by normal cells, fate of Glc in normal cells, Glc uptake in tumor cells, hexokinase in tumor cells, and 18F-labeled FDG positron emission tomog. (FDG-PET) are described. The role of FDG-PET in the management of cancer patients is discussed.147Krishnan, H. S.; Ma, L.; Vasdev, N.; Liang, S. H. 18F-Labeling of sensitive biomolecules for positron emission tomography. Chem. - Eur. J. 2017, 23, 15553– 15577, DOI: 10.1002/chem.201701581[Crossref], [PubMed], [CAS], Google Scholar147https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVekt7vP&md5=36ab8ca2d515f0b53ca9ba3eb361b55318F-Labeling of Sensitive Biomolecules for Positron Emission TomographyKrishnan, Hema S.; Ma, Longle; Vasdev, Neil; Liang, Steven H.Chemistry - A European Journal (2017), 23 (62), 15553-15577CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Positron emission tomog. (PET) imaging study of fluorine-18 labeled biomols. is an emerging and rapidly growing area for preclin. and clin. research. The present review focuses on recent advances in radiochem. methods for incorporating fluorine-18 into biomols. via "direct" or "indirect" bioconjugation. Recently developed prosthetic groups and pre-targeting strategies, as well as representative examples in 18F-labeling of biomols. in PET imaging research studies are highlighted.148Lee, K. C.; Lee, S.-Y.; Choe, Y. S.; Chi, D. Y. Metabolic stability of [18F]fluoroalkylbiphenyls. Bull. Korean Chem. Soc. 2004, 25, 1225– 1230, DOI: 10.5012/bkcs.2004.25.8.1225[Crossref], [CAS], Google Scholar148https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXosVKlu7c%253D&md5=be34e3fc985b636ca73833f435ad0204Metabolic stability of [18F]fluoroalkylbiphenylsLee, Kyo Chul; Lee, Sang-Yoon; Choe, Yearn Seong; Chi, Dae YoonBulletin of the Korean Chemical Society (2004), 25 (8), 1225-1230CODEN: BKCSDE; ISSN:0253-2964. (Korean Chemical Society)The stability of fluoroalkyl groups as a pendent on the Ph ring was measured in vitro using rat hepatic microsomes and human serum to predict their in vivo stabilities. We have prepd. three [18F]fluoroalkylbiphenyls as the model compds. of fluoroalkyl arom. compds. to compare the in vitro stabilities. In addn., in vitro stabilities were measured sep. using rat hepatic microsomes and human serum at 37°. Fluoroethylbiphenyl had similar or slightly superior stability to fluoropropylbiphenyl and these two compds. were much more stable than fluoromethylbiphenyl in vitro.149French, A. N.; Napolitano, E.; VanBrocklin, H. F.; Hanson, R. N.; Welch, M. J.; Katzenellenbogen, J. A. Synthesis, radiolabeling and tissue distribution of 11 β-fluoroalkyl- and 11 β-fluoroalkoxy-substituted estrogens: target tissue uptake selectivity and defluorination of a homologous series of fluorine-18-labeled estrogens. Nucl. Med. Biol. 1993, 20, 31– 47, DOI: 10.1016/0969-8051(93)90134-G[Crossref], [PubMed], [CAS], Google Scholar149https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXitVylsr0%253D&md5=51479b76bbfb5f044f99b6ef632d9250Synthesis, radiolabeling and tissue distribution of 11β-fluoroalkyl- and 11β-fluoroalkoxy-substituted estrogens: target tissue uptake selectivity and defluorination of a homologous series of fluorine-18-labeled estrogensFrench, Andrew N.; Napolitano, Elio; Vanbrocklin, Henry F.; Hanson, Robert N.; Welch, Michael J.; Katzenellenbogen, John A.Nuclear Medicine and Biology (1993), 20 (1), 31-47CODEN: NMBIEO; ISSN:0883-2897.Six estrogens substituted at the 11β-position with a fluoroalkyl or fluoroalkoxy substituent were prepd. These compds. bind to the estrogen receptor with moderate to high affinity, with the fluoroalkyl analogs being higher affinity binders than the fluoroalkoxy ones. All of these fluorine-substituted estrogens were prepd. in fluorine-18-labeled form, with high radiochem. purity and at effective specific activities (15.4-50.4 TBq/mmol; 415-1362 Ci/mmol)adequate for biodistribution studies. In immature female rats, 5 of the 6 fluoroestrogens showed selective uptake by the uterus, with uterine uptake as a percent of the injected dose per g being 4-9% at 1 h, and uterus-to-blood or uterus-to-muscle ratios being 10-40. Selective uterine uptake was eliminated by coadministration of a blocking dose of unlabeled estradiol. The only compd. that did not show selective uterine uptake was 11β-fluoropropoxyl estradiol; its rapid metab. and its low affinity for the estrogen receptor, particularly at 25°, may account for its lack of specific uptake. The level of bone activity, an index of metabolic defluorination, shows that the defluorination rates of these six estrogens are a complex function of structure and functionality. The least prone to defluorination is 11β-(2-fluoroethoxy)estradiol and the most prone is 11β-(2-fluoroethyl)estradiol. The extent of defluorination of the remaining compds. shows weak evidence for the protective effect of a heteroatom-substituted beta to the site of metab. (the CH bonds on the fluorine-bearing carbon atom). The binding affinity, tissue distribution and metab. of these 11β-fluoroalkyl- and fluoroalkoxy-substituted estrogens further the understanding of the behavior of fluorine-18-labeled estrogens as potential imaging agents for estrogen receptor-pos. breast cancer.150Irurre, J., Jr.; Casas, J.; Messeguer, A. Resistance of the 2,2,2-trifluoroethoxy aryl moiety to the cytochrome P-450 metabolism in rat liver microsomes. Bioorg. Med. Chem. Lett. 1993, 3, 179– 182, DOI: 10.1016/S0960-894X(01)80872-6[Crossref], [CAS], Google Scholar150https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXktVShsL8%253D&md5=3388a20b7a0ee665e8224535556eef5fResistance of the 2,2,2-trifluoroethoxy aryl moiety to the cytochrome P-450 metabolism in rat liver microsomesIrurre, Josep, Jr.; Casas, Josefina; Messeguer, AngelBioorganic & Medicinal Chemistry Letters (1993), 3 (2), 179-82CODEN: BMCLE8; ISSN:0960-894X.7-(2,2,2-Trifluoroethoxy)coumarin (I) or 4'-(2,2,2-trifluoroethoxy)acetanilide (II), fluorinated analogs of the well known cytochrome P 450 deethylase (EC 1.14.14.1) substrates 7-ethoxycoumarin and phenacetin, resp., remained unaltered after incubation with rat liver microsomes in the presence of NADPH. In addn., compds. I and II showed a moderate activity as inhibitors of the above enzymes. These results suggest that the CF3CH2O- group could play an important role in the design of bioactive compds. when a metabolic resistance at a specific position is desired.151Diana, G. D.; Rudewicz, P.; Pevear, D. C.; Nitz, T. J.; Aldous, S. C.; Aldous, D. J.; Robinson, D. T.; Draper, T.; Dutko, F. J.; Aldi, C.; Gendron, G.; Oglesby, R. C.; Volkots, D. L.; Reumann, M.; Bailey, T. R.; Czernial, R.; Block, T.; Roland, R.; Opperman, J. Picornavirus inhibitors: trifluoromethyl substitution provides a global protective effect against hepatic metabolism. J. Med. Chem. 1995, 38, 1355– 1371, DOI: 10.1021/jm00008a014[ACS Full Text], [CAS], Google Scholar151https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXkvVWqtLs%253D&md5=36a8ac2604f10f57d2aad9b131d84734Picornavirus Inhibitors: Trifluoromethyl Substitution Provides a Global Protective Effect against Hepatic MetabolismDiana, Guy D.; Rudewicz, Patrick; Pevear, Daniel C.; Nitz, Theodore J.; Aldous, Suzanne C.; Aldous, David J.; Robinson, David T.; Draper, Tandy; Dutko, Frank J.; et al.Journal of Medicinal Chemistry (1995), 38 (8), 1355-71CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Several modifications of the oxazoline ring of WIN 54954, a broad spectrum antipicornavirus compd., have been prepd. to address the acid lability and metabolic instability of this compd. The authors have previously shown that the oxadiazole analog (I) displayed comparable activity against a variety of rhinoviruses and appeared to be stable to acid. A monkey liver microsomal assay was developed to examine the metabolic stability in vitro of both compds., and it was detd. that WIN 54954 displayed 18 metabolic products while I was converted to 8 products. Two major products of I were detd. by LC-MS/MS to be monohydroxylated at each of the terminal Me groups. Replacement of the Me on the isoxazole ring with a trifluoromethyl group, while preventing hydroxylation at this position, did not reduce the sensitivity of the mol. to microsomal metab. at other sites. However, the (trifluoromethyl)oxadiazole not only prevented hydroxylation at this position but also provided protection at the isoxazole end of the mol., resulting in only two minor products to the extent of 4%. The major product was identified as the monohydroxylated compd. The global metabolic protective effect of trifluoromethyl group on the oxadiazole ring was further demonstrated by examg. a variety of analogous including heterocyclic replacements of the isoxazole ring. In each case, the trifluoromethyl analog displayed a protective effect when compared to the corresponding Me analog.152Cliffe, I. A. A retrospect on the discovery of WAY-100635 and the prospect for improved 5-HT(1A) receptor PET radioligands. Nucl. Med. Biol. 2000, 27, 441– 447, DOI: 10.1016/S0969-8051(00)00109-8[Crossref], [PubMed], [CAS], Google Scholar152https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXmtVGjt7k%253D&md5=b65fa42e0685aeb1ca0e3e0e6344402dA retrospect on the discovery of way-100635 and the prospect for improved 5-HT1A receptor PET radioligandsCliffe, I. A.Nuclear Medicine and Biology (2000), 27 (5), 441-447CODEN: NMBIEO; ISSN:0969-8051. (Elsevier Science Inc.)A review with 45 refs. An outline is given of the developments that led to the identification of [O-methyl-11C]WAY-100635 (4) as the first useful PET ligand for imaging serotonin1A (5-HT1A) receptors in the living human brain. Recent attempts to develop 5-HT1A receptor radioligands superior to 4 are reviewed, and [carbonyl-11C]WAY-100635 (6) has been shown to be the best currently available radioligand for human studies. Of other 11C-radiolabeled compds., [O-methyl-11C](R,S)-CPC-222 (9), DWAY (8), and [11C]NAD-299 (14) all demonstrate specific binding to 5-HT1A receptors in animals and warrant further expedited studies in humans. The trans-fluorocyclohexane, 12, and fluorobenzene, [18F]p-MPPF 13, are highlighted as examples of promising 18F-labeled ligands.153Pike, V. W.; Halldin, C.; Wikstrom, H.; Marchais, S.; McCarron, J. A.; Sandell, J.; Nowicki, B.; Swahn, C. G.; Osman, S.; Hume, S. P.; Constantinou, M.; Andree, B.; Farde, L. Radioligands for the study of brain 5-HT(1A) receptors in vivo--development of some new analogues of way. Nucl. Med. Biol. 2000, 27, 449– 455, DOI: 10.1016/S0969-8051(00)00110-4[Crossref], [PubMed], [CAS], Google Scholar153https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXmtVGjt7Y%253D&md5=f113308beeb2ea0ee186052477101f45Radioligands for the study of brain 5-HT1A receptors: in vivo development of some new analogues of WAYPike, V. W.; Halldin, C.; Wikstrom, H.; Marchais, S.; McCarron, J. A.; Sandell, J.; Nowicki, B.; Swahn, C.-G.; Osman, S.; Hume, S. P.; Constantinou, M.; Andree, B.; Farde, L.Nuclear Medicine and Biology (2000), 27 (5), 449-455CODEN: NMBIEO; ISSN:0969-8051. (Elsevier Science Inc.)A review with 46 refs. [Carbonyl-11C]WAY-100635 (WAY) has proved to be a very useful radioligand for the imaging of brain 5-HT1A receptors in human brain in vivo with positron emission tomog. (PET). WAY is now being applied widely for clin. research and drug development. However, WAY is rapidly cleared from plasma and is also rapidly metabolized. A comparable radioligand, with a higher and more sustained delivery to brain, is desirable since these properties might lead to better biomathematical modeling of acquired PET data. There are also needs for other types of 5-HT1A receptor radioligands, for example, ligands sensitive to elevated serotonin levels, ligands labeled with longer-lived fluorine-18 for distribution to "satellite" PET centers, and ligands labeled with iodine-123 for single photon emission computerized tomog. (SPECT) imaging. Here we describe our progress toward these aims through the exploration of WAY analogs, including the development of [carbonyl-11C]desmethyl-WAY (DWAY) as a promising, more brain-penetrant radioligand for PET imaging of human 5-HT1A receptors, and (pyridinyl-6-halo)-analogs as promising leads for the development of radiohalogenated ligands.154Al Hussainy, R.; Verbeek, J.; van der Born, D.; Molthoff, C.; Booij, J.; Herscheid, J. K. Synthesis, biodistribution and PET studies in rats of 18F-labeled bridgehead fluoromethyl analogues of WAY-100635. Nucl. Med. Biol. 2012, 39, 1068– 1076, DOI: 10.1016/j.nucmedbio.2012.04.002[Crossref], [PubMed], [CAS], Google Scholar154https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhtlamu7nM&md5=488c8af356c6fd2550723321be5bcb7eSynthesis, biodistribution and PET studies in rats of 18F-Labeled bridgehead fluoromethyl analogues of WAY-100635Al Hussainy, Rana; Verbeek, Joost; van der Born, Dion; Molthoff, Carla; Booij, Jan; Herscheid, J. D. M.Nuclear Medicine and Biology (2012), 39 (7), 1068-1076CODEN: NMBIEO; ISSN:0969-8051. (Elsevier)In vitro screening of fluoromethyl bridge-fused ring (BFR) analogs of WAY-100635 (5a, 5b and 5c) has shown a high binding affinity and a good selectivity for the 5-HT1A receptor. As these compds. were designed to provide PET ligands with high metabolic stability, they are now radiolabeled with fluorine-18 and investigated in vivo. BFR precursors were synthesized and reacted with fluorine-18 in dry MeCN in the presence of 2,2,2-kryptofix and K2CO3. In rats, biodistribution and PET studies were performed using [18F]5a, [18F]5b and [18F]5c. The binding specificity was detd. by administration of non-labeled WAY-100635 prior to the radiolabeled ligands. [18F]5 ligands were synthesized in overall radiochem. yields of 24%-45%, resp. with a radiochem. purity of > 98%. Relatively good hippocampus to cerebellum ratios of 5.55, 4.79 and 5.45, resp. were reached at 45 min pi. However, PET studies indicated defluorination of the radioligands by showing high accumulation of radioactivity in the bones in the order of [18F]5a ≈ [18F]5b > [18F]5c. Also in vivo, the radioligands bind preferentially to the 5-HT1A receptor. Unfortunately, no metabolic stability with regard to defluorination was obsd. in rats.155Saigal, N.; Pichika, R.; Easwaramoorthy, B.; Collins, D.; Christian, B. T.; Shi, B.; Narayanan, T. K.; Potkin, S. G.; Mukherjee, J. Synthesis and biologic evaluation of a novel serotonin 5-HT1A receptor radioligand, 18F-labeled mefway, in rodents and imaging by PET in a nonhuman primate. J. Nucl. Med. 2006, 47, 1697– 1706[PubMed], [CAS], Google Scholar155https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFKjtrbL&md5=69b747cf192da90a097b5c27db14051eSynthesis and biologic evaluation of a novel serotonin 5-HT1A receptor radioligand, 18F-labeled mefway, in rodents and imaging by PET in a nonhuman primateSaigal, Neil; Pichika, Rama; Easwaramoorthy, Balasubramaniam; Collins, Daphne; Christian, Bradley T.; Shi, Bingzhi; Narayanan, Tanjore K.; Potkin, Steven G.; Mukherjee, JogeshwarJournal of Nuclear Medicine (2006), 47 (10), 1697-1706CODEN: JNMEAQ; ISSN:0161-5505. (Society of Nuclear Medicine)Serotonin 5-HT1A receptors have been implicated in disorders of the central nervous system and, therefore, are being studied by PET. Efforts are under way to improve in vivo stability of 5-HT1A agents currently in human use (11C-labeled N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-(2-pyridinyl)cyclohexanecarboxamide [11C-WAY-100635]), 4-(2'-methoxyphenyl)-1-[2'-(N-2''-pyridinyl)-p-18F-fluorobenzamido]ethylpiperazine [18F-MPPF], and 18F-labeled trans-4-fluoro-N-(2-[4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-N-(2-pyridyl)cyclohexanecarboxamide) [18F-FCWAY]. We have synthesized N-{2-[4-(2-methoxyphenyl)piperazinyl]ethyl}-N-(2-pyridyl)-N-(4-18F-fluoromethylcyclohexane)carboxamide (18F-mefway), which contains a 18F on a primary carbon to make the compd. more stable to defluorination. Methods: Radiosynthesis of 18F-mefway was performed in a single tosylate for 18F-fluoride exchange. In vitro binding studies on rat brain slices using 18F-mefway were read on a phosphor imager. Monkey PET studies were performed on a whole-body PET scanner. Results: Binding affinity (inhibitory concn. of 50% [IC50]) of mefway was 26 nmol/L and was comparable to that of WAY-100635, 23 nmol/L. Yields of 18F-mefway were 20%-30% in specific activities of 74-111 GBq/μmol at the end of synthesis. In vitro binding of 18F-mefway in the hippocampus (Hp), colliculus (Co), cortex (Ctx), and other brain regions-with limited binding in the cerebellum (Cer)-was obsd., with ratios of Hp/Cer = 82.3, Co/Cer = 45.8, and Ctx/Cer = 40. Serotonin displaced 18F-mefway from various brain regions with IC50 values in the range of 169-243 nmol/L. PET studies in a rhesus monkey showed 18F-mefway binding in the fontal cortex (FC), temporal cortex (TC) including hippocampus, raphe (Rp), and other brain regions, with ratios of FC/Cer = 9.0, TC/Cer = 10, and Rp/Cer = 3.3. Plasma anal. indicated the presence of approx. 30% of 18F-mefway at 150-180 min after injection. Conclusion: The high ratios in specific brain regions such as the hippocampus suggest that 18F-mefway has potential as a PET agent for 5HT1A receptors.156Rodil, A.; Bosisio, S.; Ayoup, M. S.; Quinn, L.; Cordes, D. B.; Slawin, A. M. Z.; Murphy, C. D.; Michel, J.; O’Hagan, D. Metabolism and hydrophilicity of the polarised ‘Janus face’ all-cis tetrafluorocyclohexyl ring, a candidate motif for drug discovery. Chem. Sci. 2018, 9, 3023– 3028, DOI: 10.1039/C8SC00299A[Crossref], [PubMed], [CAS], Google Scholar156https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXivFSmsr8%253D&md5=d6b676dd05116c6a9e1c39093f415fabMetabolism and hydrophilicity of the polarised 'Janus face' all-cis tetrafluorocyclohexyl ring, a candidate motif for drug discoveryRodil, Andrea; Bosisio, Stefano; Ayoup, Mohammed Salah; Quinn, Laura; Cordes, David B.; Slawin, Alexandra M. Z.; Murphy, Cormac D.; Michel, Julien; O'Hagan, DavidChemical Science (2018), 9 (11), 3023-3028CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)The metab. and polarity of the all-cis tetra-fluorocyclohexane motif is explored in the context of its potential as a motif for inclusion in drug discovery programs. Biotransformations of Ph all-cis tetra-, tri- and di- fluoro cyclohexanes with the human metab. model organism Cunninghamella elegans illustrates various hydroxylated products, but limited to benzylic hydroxylation for the Ph all-cis tetrafluorocyclohexyl ring system. Evaluation of the lipophilicities (log P) indicates a significant and progressive increase in polarity with increasing fluorination on the cyclohexane ring system. Mol. dynamics simulations indicate that water assocs. much more closely with the hydrogen face of these Janus face cyclohexyl rings than the fluorine face owing to enhanced hydrogen bonding interactions with the polarised hydrogens and water.157Harlow, P. H.; Perry, S. J.; Stevens, A. J.; Flemming, A. J. Comparative metabolism of xenobiotic chemicals by cytochrome P450s in the nematode Caenorhabditis elegans. Sci. Rep. 2018, 8, 13333, DOI: 10.1038/s41598-018-31215-w[Crossref], [PubMed], [CAS], Google Scholar157https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3c3mtlCktg%253D%253D&md5=85e0c599a138cbdacc9794815095d92eComparative metabolism of xenobiotic chemicals by cytochrome P450s in the nematode Caenorhabditis elegansHarlow Philippa H; Perry Simon J; Stevens Alexander J; Flemming Anthony JScientific reports (2018), 8 (1), 13333 ISSN:.We investigated the metabolic capabilities of C. elegans using compounds whose metabolism has been well characterised in mammalian systems. We find that similar metabolites are produced in C. elegans as in mammals but that C. elegans is deficient in CYP1-like metabolism, as has been seen in other studies. We show that CYP-34A9, CYP-34A10 and CYP-36A1 are the principal enzymes responsible for the metabolism of tolbutamide in C. elegans. These are related to the mammalian enzymes that metabolise this compound but are not the closest homologs suggesting that sequence comparison alone will not predict functional conservation among cytochrome P450s. In mammals, metabolite production from amytryptiline and dextromethorphan is dependent on specific cytochrome P450s. However, in C. elegans we did not find evidence of similar specificity: the same metabolites were produced but in small amounts by numerous cytochrome P450s. We conclude that, while some aspects of cytochrome P450 mediated metabolism in C. elegans are similar to mammals, there are differences in the production of some metabolites and in the underlying genetics of metabolism.158Huchet, Q. A.; Kuhn, B.; Wagner, B.; Kratochwil, N. A.; Fischer, H.; Kansy, M.; Zimmerli, D.; Carreira, E. M.; Muller, K. Fluorination patterning: A study of structural motifs that impact physicochemical properties of relevance to drug discovery. J. Med. Chem. 2015, 58, 9041– 9060, DOI: 10.1021/acs.jmedchem.5b01455[ACS Full Text], [CAS], Google Scholar158https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhslGmsrzM&md5=20d7a36b75501c02570744857c82f325Fluorination Patterning: A Study of Structural Motifs That Impact Physicochemical Properties of Relevance to Drug DiscoveryHuchet, Quentin A.; Kuhn, Bernd; Wagner, Bjorn; Kratochwil, Nicole A.; Fischer, Holger; Kansy, Manfred; Zimmerli, Daniel; Carreira, Erick M.; Muller, KlausJournal of Medicinal Chemistry (2015), 58 (22), 9041-9060CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The synthesis of a collection of 3-substituted indole derivs. incorporating partially fluorinated Pr and Bu groups is described along with an in-depth study of the effects of various fluorination patterns on their properties, such as lipophilicity, aq. soly., and metabolic stability. The exptl. observations confirm predictions of a marked lipophilicity decrease imparted by a vic-difluoro unit when compared to the gem-difluoro counterparts. The data involving the comparison of the two substitution patterns is expected to benefit mol. design in medicinal chem. and, more broadly, in life as well as materials sciences.159Xie, C.; Gao, X.; Sun, D.; Zhang, Y.; Krausz, K. W.; Qin, X.; Gonzalez, F. J. Metabolic profiling of the novel hypoxia-inducible factor 2α inhibitor PT2385 in vivo and In vitro. Drug Metab. Dispos. 2018, 46, 336– 345, DOI: 10.1124/dmd.117.079723[Crossref], [PubMed], [CAS], Google Scholar159https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpsV2ktL8%253D&md5=c1c2ac8c068be824926381074cc046daMetabolic profiling of the novel hypoxia-inducible factor 2a inhibitor PT2385 in vivo and in vitroXie, Cen; Gao, Xiaoxia; Sun, Dongxue; Zhang, Youbo; Krausz, Kristopher W.; Qin, Xuemei; Gonzalez, Frank J.Drug Metabolism & Disposition (2018), 46 (4), 336-345,S1-S14CODEN: DMDSAI; ISSN:1521-009X. (American Society for Pharmacology and Experimental Therapeutics)PT2385 is a first-in-class, selective small-mol. inhibitor of hypoxia-inducible factor-2α (HIF-2α) developed for the treatment of advanced clear cell renal cell carcinoma. Preclin. results demonstrated that PT2385 has potent antitumor efficacy in mouse xenograft models of kidney cancer. It also has activity toward metabolic disease in a mouse model. However, no metab. data are currently publically available. It is of great importance to characterize the metab. of PT2385 and identify its effect on systemic homeostasis in mice. High-resoln. mass spectrometry-based metabolomics was performed to profile the biotransformation of PT2385 and PT2385-induced changes in endogenous metabolites. Liver microsomes and recombinant drug-metabolizing enzymes were used to det. the mechanism of PT2385 metab. Real-time polymerase chain reaction anal. was employed to investigate the reason for the PT2385-induced bile acid dysregulation. A total of 12 metabolites of PT2385 was characterized, generated from hydroxylation (M1, M2), dihydroxylation and desatn. (M3, M4), oxidative-defluorination (M7), glucuronidation (M8), N-acetylcysteine conjugation (M9), and secondary methylation (M5, M6) and glucuronidation (M10, M11, and M12). CYP2C19 was the major contributor to the formation of M1, M2, and M7, UGT2B17 to M8, and UGT1A1/3 to M10-M12. The bile acid metabolites taurocholic acid and tauro-β-muricholic acid were elevated in serum and liver of mice after PT2385 treatment. Gene expression anal. further revealed that intestinal HIF-2α inhibition by PT2385 treatment upregulated the hepatic expression of CYP7A1, the rate-limiting enzyme in bile acid synthesis. This study provides metabolic data and an important ref. basis for the safety evaluation and rational clin. application of PT2385.160Xu, R.; Wang, K.; Rizzi, J. P.; Huang, H.; Grina, J. A.; Schlachter, S. T.; Wang, B.; Wehn, P. M.; Yang, H.; Dixon, D. D.; Czerwinski, R. M.; Du, X.; Ged, E. L.; Han, G.; Tan, H.; Wong, T.; Xie, S.; Josey, J. A.; Wallace, E. M. 3-[(1S,2S,3R)-2,3-Difluoro-1-hydroxy-7-methylsulfonylindan-4-yl]oxy-5-fluorobenzo nitrile (PT2977), a hypoxia-inducible factor 2α (HIF-2α) inhibitor for the treatment of clear cell renal cell carcinoma. J. Med. Chem. 2019, 62, 6876– 6893, DOI: 10.1021/acs.jmedchem.9b00719[ACS Full Text], [CAS], Google Scholar160https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1ensL%252FN&md5=ec1f0a964e0fda36f92261c30edf284e3-[(1S,2S,3R)-2,3-Difluoro-1-hydroxy-7-methylsulfonylindan-4-yl]oxy-5-fluorobenzonitrile (PT2977), a Hypoxia-Inducible Factor 2α (HIF-2α) Inhibitor for the Treatment of Clear Cell Renal Cell CarcinomaXu, Rui; Wang, Keshi; Rizzi, James P.; Huang, Heli; Grina, Jonas A.; Schlachter, Stephen T.; Wang, Bin; Wehn, Paul M.; Yang, Hanbiao; Dixon, Darryl D.; Czerwinski, Robert M.; Du, Xinlin; Ged, Emily L.; Han, Guangzhou; Tan, Huiling; Wong, Tai; Xie, Shanhai; Josey, John A.; Wallace, Eli M.Journal of Medicinal Chemistry (2019), 62 (15), 6876-6893CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The hypoxia-inducible factor 2α (HIF-2α) is a key oncogenic driver in clear cell renal cell carcinoma (ccRCC). Our first HIF-2α inhibitor PT2385 demonstrated promising proof of concept clin. activity in heavily pretreated advanced ccRCC patients. However, PT2385 was restricted by variable and dose-limited pharmacokinetics resulting from extensive metab. of PT2385 to its glucuronide metabolite. Herein we describe the discovery of second-generation HIF-2α inhibitor PT2977 with increased potency and improved pharmacokinetic profile achieved by redn. of phase 2 metab. Structural modification by changing the geminal difluoro group in PT2385 to a vicinal difluoro group resulted in enhanced potency, decreased lipophilicity, and significantly improved pharmacokinetic properties. In a phase 1 dose-escalation study, the clin. pharmacokinetics for PT2977 supports the hypothesis that attenuating the rate of glucuronidation would improve exposure and reduce variability in patients. Early evidence of clin. activity shows promise for PT2977 in the treatment of ccRCC.161Hughes, S. C.; Shardlow, P. C.; Hollis, F. J.; Scott, R. J.; Motivaras, D. S.; Allen, A.; Rousell, V. M. Metabolism and disposition of fluticasone furoate, an enhanced-affinity glucocorticoid, in humans. Drug Metab. Dispos. 2008, 36, 2337– 2344, DOI: 10.1124/dmd.108.022137[Crossref], [PubMed], [CAS], Google Scholar161https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtlWiu77L&md5=d51c628e6fdc783fa5fd8dc58d4cdaa6Metabolism and disposition of fluticasone furoate, an enhanced-affinity glucocorticoid, in humansHughes, Stephen C.; Shardlow, Peter C.; Hollis, Frank J.; Scott, Rebecca J.; Motivaras, Dimple S.; Allen, Ann; Rousell, Victoria M.Drug Metabolism and Disposition (2008), 36 (11), 2337-2344CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)The purpose of this study was to investigate the metab. and disposition of fluticasone furoate, an enhanced-affinity glucocorticoid receptor agonist, in humans. In a two-part, open-label design study, five healthy male subjects received a p.o. dose of 2 mg of [14C]fluticasone furoate, followed 4 wk later by an i.v. dose of 0.25 mg of [14C]fluticasone furoate (as a 30-min infusion). Oral absorption was rapid and estd. at approx. 30%, although the oral bioavailability was markedly lower at 1.6%, limited by extensive first-pass metab. Plasma clearance was 58.3 L/h, with a vol. of distribution of 642 L and a terminal elimination half-life of 15.3 h. The major circulating component identified in plasma exts. after i.v. and p.o. dosing was unchanged parent compd., with 17β-carboxylic acid (GW 694301X) also being notable after p.o. administration. Mean recovery of radioactivity was approx. 92 and 102% at 216 and 168 h after i.v. and p.o. administration, resp., with most (at least 90%) recovered in the feces. Fluticasone furoate was extensively metabolized, with only trace amts. of unchanged parent compd. obsd. in feces following either route of administration. The predominant pathway was removal of the S-fluoromethyl carbothioate group to yield GW 694301X. Other pathways included oxidative defluorination to yield a hydroxyl at the C6 position. There was no evidence for metabolic loss of the furoate group from fluticasone furoate or any of its metabolites. Evidence presented suggests that enterocytes have a role in the metab. of unabsorbed fluticasone furoate.162Teitelbaum, P. J.; Chu, N. I.; Cho, D.; Tökés, L.; Patterson, J. W.; Wagner, P. J.; Chaplin, M. D. Mechanism for the oxidative defluorination of flunisolide. J. Pharmacol. Exp. Ther. 1981, 218, 16– 22[PubMed], [CAS], Google Scholar162https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXkslCisL8%253D&md5=5aeb5f0bda2f31d237bfccd176025878Mechanism for the oxidative defluorination of flunisolideTeitelbaum, Philip J.; Chu, Nancy I.; Cho, Diane; Toekes, Laszlo; Patterson, John W.; Wagner, Paul J.; Chaplin, Melvin D.Journal of Pharmacology and Experimental Therapeutics (1981), 218 (1), 16-22CODEN: JPETAB; ISSN:0022-3565.Flunisolide (I) [3385-03-3] was converted to 6β,11β,16α,17α,21-pentahydroxypregna-1,4-diene-3,20-dione-16,17-acetonide (6β-OH metabolite) [73603-90-4] by mouse liver microsomes, but no activity was obsd. with mouse lung, intestine or kidney microsomes. Two addnl. metabolites of I also formed by mouse hepatic microsomes were identified by mass spectral anal. to be 11β,16α,17α,21-tetrahydroxypregna-1,4-diene-3,6,20-trione 16,17-acetonide (6-keto metabolite) [78228-98-5] and Δ6-flunisolide [78245-15-5]. The formation of all 3 metabolites required NADPH [53-57-6], was inhibited by CO, and was stimulated by pretreating mice with phenobarbital. A time-course study suggested the 6-keto metabolite was an intermediate in the formation of the 6β-OH metabolite. When added to microsomes, the 6-keto metabolite was converted to the 6β-OH metabolite by a CO-insensitive enzyme. Apparently, the conversion of I to the 6β-OH metabolite is catalyzed by a multi-enzyme pathway via a stable intermediate, the 6-keto metabolite. The initial reaction which leads to the formation of the 6-keto metabolite is catalyzed by a cytochrome P-450-mediated microsomal monoxygenase(s), but the redn. of the 6-keto metabolite to the 6β-OH metabolite is cytochrome P-450-independent.163Moore, C. D.; Roberts, J. K.; Orton, C. R.; Murai, T.; Fidler, T. P.; Reilly, C. A.; Ward, R. M.; Yost, G. S. Metabolic pathways of inhaled glucocorticoids by the CYP3A enzymes. Drug Metab. Dispos. 2013, 41, 379– 389, DOI: 10.1124/dmd.112.046318[Crossref], [PubMed], [CAS], Google Scholar163https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXitlGis7k%253D&md5=e89d33f0da338a12b5258e543ae6234fMetabolic pathways of inhaled glucocorticoids by the CYP3A enzymesMoore, Chad D.; Roberts, Jessica K.; Orton, Christopher R.; Murai, Takahiro; Fidler, Trevor P.; Reilly, Christopher A.; Ward, Robert M.; Yost, Garold S.Drug Metabolism & Disposition (2013), 41 (2), 379-389CODEN: DMDSAI; ISSN:1521-009X. (American Society for Pharmacology and Experimental Therapeutics)Asthma is one of the most prevalent diseases in the world, for which the mainstay treatment has been inhaled glucocorticoids (GCs). Despite the widespread use of these drugs, approx. 30% of asthma sufferers exhibit some degree of steroid insensitivity or are refractory to inhaled GCs. One hypothesis to explain this phenomenon is interpatient variability in the clearance of these compds. The objective of this research is to det. how metab. of GCs by the CYP3A family of enzymes could affect their effectiveness in asthmatic patients. In this work, the metab. of four frequently prescribed inhaled GCs, triamcinolone acetonide, flunisolide, budesonide, and fluticasone propionate, by the CYP3A family of enzymes was studied to identify differences in their rates of clearance and to identify their metabolites. Both interenzyme and interdrug variability in rates of metab. and metabolic fate were obsd. CYP3A4 was the most efficient metabolic catalyst for all the compds., and CYP3A7 had the slowest rates. CYP3A5, which is particularly relevant to GC metab. in the lungs, was also shown to efficiently metabolize triamcinolone acetonide, budesonide, and fluticasone propionate. In contrast, flunisolide was only metabolized via CYP3A4, with no significant turnover by CYP3A5 or CYP3A7. Common metabolites included 6β-hydroxylation and Δ6-dehydrogenation for triamcinolone acetonide, budesonide, and flunisolide. The structure of Δ6-flunisolide was unambiguously established by NMR anal. Metab. also occurred on the D-ring substituents, including the 21-carboxy metabolites for triamcinolone acetonide and flunisolide. The novel metabolite 21-nortriamcinolone acetonide was also identified by liq. chromatog.-mass spectrometry and NMR anal.164Biggadike, K.; Bledsoe, R. K.; Hassell, A. M.; Kirk, B. E.; McLay, I. M.; Shewchuk, L. M.; Stewart, E. L. X-ray crystal structure of the novel enhanced-affinity glucocorticoid agonist fluticasone furoate in the glucocorticoid receptor-ligand binding domain. J. Med. Chem. 2008, 51, 3349– 3352, DOI: 10.1021/jm800279t[ACS Full Text], [CAS], Google Scholar164https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXms1Gis7w%253D&md5=bb9838014ade0fbe90c0693b3a8191eaX-ray Crystal Structure of the Novel Enhanced-Affinity Glucocorticoid Agonist Fluticasone Furoate in the Glucocorticoid Receptor-Ligand Binding DomainBiggadike, Keith; Bledsoe, Randy K.; Hassell, Anne M.; Kirk, Barrie E.; McLay, Iain M.; Shewchuk, Lisa M.; Stewart, Eugene L.Journal of Medicinal Chemistry (2008), 51 (12), 3349-3352CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)An X-ray crystal structure is reported for the novel enhanced-affinity glucocorticoid agonist fluticasone furoate (FF) in the ligand binding domain of the glucocorticoid receptor. Comparison of this structure with those of dexamethasone and fluticasone propionate shows the 17α furoate ester to occupy more fully the lipophilic 17α pocket on the receptor, which may account for the enhanced glucocorticoid receptor binding of FF.165Krauser, J. A.; Guengerich, F. P. Cytochrome P450 3A4-catalyzed testosterone 6β-hydroxylation stereochemistry, kinetic deuterium isotope effects, and rate-limiting steps. J. Biol. Chem. 2005, 280, 19496– 19506, DOI: 10.1074/jbc.M501854200[Crossref], [PubMed], [CAS], Google Scholar165https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXktFymsLY%253D&md5=96020ed1c61b61e6147e30b793e39f04Cytochrome P450 3A4-catalyzed Testosterone 6β-Hydroxylation Stereochemistry, Kinetic Deuterium Isotope Effects, and Rate-limiting StepsKrauser, Joel A.; Guengerich, F. PeterJournal of Biological Chemistry (2005), 280 (20), 19496-19506CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Testosterone 6β-hydroxylation is a prototypic reaction of cytochrome P 450 (P 450) 3A4, the major human P 450. Biomimetic reactions produced a variety of testosterone oxidn. products with 6β-hydroxylation being only a minor reaction, indicating that P 450 3A4 has considerable control over the course of steroid hydroxylation because 6β-hydroxylation is not dominant in a thermodynamically controlled oxidn. of the substrate. Several isotopically labeled testosterone substrates were prepd. and used to probe the catalytic mechanism of P 450 3A4: (1) 2,2,4,6,6-2H5; (2) 6,6-2H2; (3) 6α-2H; (4) 6β-2H; and (5) 6β-3H testosterone. Only the 6β-hydrogen was removed by P 450 3A4 and not the 6α, indicating that P 450 3A4 abstrs. hydrogen and rebounds oxygen only at the β face. Anal. of the rates of hydroxylation of 6β-1H-, 6β-2H-, and 6β-3H-labeled testosterone and application of the Northrop method yielded an apparent intrinsic kinetic deuterium isotope effect (Dk) of 15. The deuterium isotope effects on kcat and kcat/Km in non-competitive reactions were only 2-3. Some switching to other hydroxylations occurred because of 6β-2H substitution. The high Dk value is consistent with an initial hydrogen atom abstraction reaction. Attenuation of the high Dk in the non-competitive expts. implies that C-H bond breaking is not a dominant rate-limiting step. Considerable attenuation of a high Dk value was also seen with a slower P 450 3A4 reaction, the O-dealkylation of 7-benzyloxyquinoline. Thus P 450 3A4 is an enzyme with regioselective flexibility but also considerable regioselectivity and stereoselectivity in product formation, not necessarily dominated by the ease of C-H bond breaking.166Liu, X.; Poddar, S.; Song, L.; Hendrickson, H.; Zhang, X.; Yuan, Y.; Zhou, D.; Zheng, G. Synthesis and liver microsomal metabolic stability studies of a fluoro-substituted δ-tocotrienol derivative. ChemMedChem 2020, DOI: 10.1002/cmdc.201900676167Chang, W.; Mosley, R. T.; Bansal, S.; Keilman, M.; Lam, A. M.; Furman, P. A.; Otto, M. J.; Sofia, M. J. Inhibition of hepatitis C virus NS5A by fluoro-olefin based γ-turn mimetics. Bioorg. Med. Chem. Lett. 2012, 22, 2938– 2942, DOI: 10.1016/j.bmcl.2012.02.051[Crossref], [PubMed], [CAS], Google Scholar167https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XktV2qtLk%253D&md5=513fc896ce045cbf1a236d75bb75b29cInhibition of hepatitis C virus NS5A by fluoro-olefin based γ-turn mimeticsChang, Wonsuk; Mosley, Ralph T.; Bansal, Shalini; Keilman, Meg; Lam, Angela M.; Furman, Phillip A.; Otto, Michael J.; Sofia, Michael J.Bioorganic & Medicinal Chemistry Letters (2012), 22 (8), 2938-2942CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)The HCV non-structural protein NS5A has been established as a viable target for the development of direct acting antiviral therapy. From computational modeling studies strong intra-mol. hydrogen bonds were a common structural moiety within known NS5A inhibitors that have low pico-molar replicon potency. Efforts to reproduce these γ-turn-like substructures provided a novel NS5A inhibitor based on a fluoro-olefin isostere. This fluoro-olefin contg. inhibitor exhibited picomolar activity (EC50 = 79 pM) against HCV genotype 1b replicon without measurable cytotoxicity. This level of activity is comparable to the natural peptide-based inhibitors currently under clinic evaluation, and demonstrates that a peptidomimetic approach can serve as a useful strategy to produce potent and structurally unique inhibitors of HCV NS5A.168Bartlett, P. A.; Otake, A. Fluoroalkenes as peptide isosteres: ground state analogs inhibitors of thermolysin. J. Org. 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The correlation indicates that these inhibitors bind as ground state analogs and represent the first direct assessment of the fluoroalkene unit as a peptide surrogate.169Welch, J. T.; Allmendinger, T. Fluoroolefin isosteres. Methods Mol. Med. 1998, 23, 357– 384, DOI: 10.1385/0-89603-517-4:357170Nielsen, O. J.; Javadi, M. S.; Sulbaek Andersen, M. P.; Hurley, M. D.; Wallington, T. J.; Singh, R. Atmospheric chemistry of CF3CF = CH2: Kinetics and mechanisms of gas-phase reactions with Cl atoms, OH radicals, and O3. Chem. Phys. Lett. 2007, 439, 18– 22, DOI: 10.1016/j.cplett.2007.03.053[Crossref], [CAS], Google Scholar170https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXkslWrtL8%253D&md5=e37616d9e52e32cb36d04e670ba7170dAtmospheric chemistry of CF3CF=CH2: Kinetics and mechanisms of gas-phase reactions with Cl atoms, OH radicals, and O3Nielsen, O. J.; Javadi, M. S.; Sulbaek Andersen, M. P.; Hurley, M. D.; Wallington, T. J.; Singh, R.Chemical Physics Letters (2007), 439 (1-3), 18-22CODEN: CHPLBC; ISSN:0009-2614. (Elsevier B.V.)Long path length FTIR-smog chamber techniques were used to det. k(Cl + CF3CF=CH2) = (7.03 ± 0.59) × 10-11, k(OH + CF3CF=CH2) = (1.05 ± 0.17) × 10-12, and k(O3 + CF3CF=>CH2) = (2.77 ± 0.21) × 10-21 cm3 mol.-1 s-1 in 700 Torr of N2, N2/O2, or air diluent at 296 K. CF3CF=CH2 has an atm. lifetime of approx. 11 days and a global warming potential (100 yr time horizon) of four. CF3CF=CH2 has a negligible global warming potential and will not make any significant contribution to radiative forcing of climate change.171Tang, X.; Madronich, S.; Wallington, T.; Calamari, D. Changes in tropospheric composition and air quality. J. Photochem. 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Assuming that other factors remain const., addnl. UV-B will increase the rate at which primary pollutants are removed from the troposphere. Increased UV-B is expected to increase the concn. of hydroxyl radicals and result in faster removal of pollutants such as carbon monoxide, methane, nonmethane hydrocarbons (NMHCs), sulfur oxides, nitrogen oxides, hydrochlorofluorocarbons (HCFCs), and hydrofluorocarbons (HFCs). Concns. of peroxy radicals (both inorg. and org.) are expected to increase, leading to higher atm. levels of hydrogen peroxide and org. peroxides. The effects of UV-B increases on tropospheric O3, OH, methane, CO, and possibly other tropospheric constituents, while not negligible, will be difficult to detect because the concns. of these species are also influenced by many other variable factors (e.g., emissions). Trifluoroacetic acid (TFA) is produced in the atm. by the degrdn. of HCFC-123 (CF3CHCl2), HCFC-124 (CF3CHFCl), and HFC-134a (CF3CH2F), which are used as substitutes for ozone-depleting substances. The atm. oxidn. mechanisms of these replacement compds. are well established. Reported measurements of TFA in rain, rivers, lakes, and oceans show it to be a ubiquitous component of the hydrosphere, present at levels much higher than can be explained by reported sources. The levels of TFA produced by the atm. degrdn. of HFCs and HCFCs emitted up to the year 2020 are estd. to be orders of magnitude below those of concern, and to make only a minor contribution to the current environmental burden of TFA. No significant effects on humans or the environment have been identified from TFA produced by atm. degrdn. of HCFCs and HFCs. Numerous std. short-term studies have shown that TFA has, at most, moderate toxicity.172Cook, E. W.; Pierce, J. S. Toxicology of fluoro-olefins. Nature 1973, 242, 337– 338, DOI: 10.1038/242337a0[Crossref], [PubMed], [CAS], Google Scholar172https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3sXhsVCjtrY%253D&md5=73681c1675dfb3f05bff704bb3d266e1Toxicology of fluoroolefinsCook, E. W.; Pierce, J. S.Nature (London, United Kingdom) (1973), 242 (5396), 337-8CODEN: NATUAS; ISSN:0028-0836.A review with 10 refs. The direct correlation between the toxicity of fluoroolefins and the reactivity of the olefins to nucleophiles, and the enhancement of the toxicity by olefin hydrolysis are discussed.173Timperley, C. M. Fluoroalkene chemistry. Part 1. Highly-toxic fluorobutenes and their mode of toxicity: reactions of perfluoroisobutene and polyfluorinated cyclobutenes with thiols. J. Fluorine Chem. 2004, 125, 685– 693, DOI: 10.1016/j.jfluchem.2003.11.021[Crossref], [CAS], Google Scholar173https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXjvVGgsbw%253D&md5=e73daba9789a18af94ebfcbf01f741cbFluoroalkene chemistry. Part 1. Highly-toxic fluorobutenes and their mode of toxicity: reactions of perfluoroisobutene and polyfluorinated cyclobutenes with thiolsTimperley, Christopher M.Journal of Fluorine Chemistry (2004), 125 (5), 685-693CODEN: JFLCAR; ISSN:0022-1139. (Elsevier Science B.V.)The reactions of four highly-toxic fluorobutenes - perfluoroisobutene (PFIB), 1-hydropentafluorocyclobutene (1-H), hexafluorocyclobutene (HFCB) and 3-chloropentafluorocyclobutene (3-Cl)-with propanethiol, 2,6-dimethoxybenzenethiol and N-acetylcysteine iso-Pr ester were studied. PFIB and HFCB reacted with two molar equivalents of the aliph. thiols, but with only one molar equivalent of the arom. thiol (presumably due to steric hindrance) and resembled phosgene in their reactivity. The fluorocyclobutenes 1-H and 3-Cl reacted with one and up to three molar equivalents of the aliph. thiols, resp., but with only one molar equivalent of the arom. thiol. The products of allyl and vinyl substitution were isolated and characterized as fully as possible. The inhalation toxicities of the fluorocyclobutenes to rodents correlated with the no. of easily-displaceable fluorine substituents, supporting the contention that toxicity is due to reaction with biol. thiols in the lung.174Timperley, C. M. Fluoroalkene chemistry. Part 2. Reactions of thiols with some toxic 1,2-dichlorinatedpolyfluorocycloalkenes. J. Fluorine Chem. 2004, 125, 1265– 1272, DOI: 10.1016/j.jfluchem.2004.02.009[Crossref], [CAS], Google Scholar174https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmvV2htbk%253D&md5=9dfd13f88e44585d38ef48a147005514Fluoroalkene chemistry Part 2. Reactions of thiols with some toxic 1,2-dichlorinated polyfluorocycloalkenesTimperley, Christopher M.Journal of Fluorine Chemistry (2004), 125 (9), 1265-1272CODEN: JFLCAR; ISSN:0022-1139. (Elsevier B.V.)1,2-Dichlorotetrafluorocyclobutene, 1,2-dichlorohexafluorocyclopentene and 1,2-dichlorooctafluorocyclohexene were treated with an equimolar amt. of benzenethiol, 2-methoxybenzenethiol, 3-methoxybenzenethiol and 4-methoxybenzenethiol in acetonitrile with potassium carbonate. Each combination of fluoroalkene and thiol gave a mixt. of mono and bis vinyl substitution products whose proportions depended on the ring size of the fluorocycloalkene and the size and electronic characteristics of the thiol. Treatment of 1,2-dichlorotetrafluorocyclobutene with one or two molar equivalents of N-acetylcysteine iso-Pr ester in acetonitrile with potassium carbonate produced the mono and bis vinyl substitution products accordingly. The results support the contention that the high inhalation toxicity of the fluorocycloalkenes is due to reaction with two molar equivalents of biol. thiols in the lung.175Alaaeddin, M. H.; Sapuan, S. M.; Zuhri, M. Y. M.; Zainudin, E. S.; Al-Oqla, F. M. Properties and common industrial applications of polyvinyl fluoride (PVF) and polyvinylidene fluoride (PVDF). IOP Conf. Ser.: Mater. Sci. Eng. 2018, 409, 012021, DOI: 10.1088/1757-899X/409/1/012021[Crossref], [CAS], Google Scholar175https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisFKktrnK&md5=68fc4def1f324630f9984732324a10a4Properties and common industrial applications of polyvinyl fluoride (PVF) and polyvinylidene fluoride (PVDF)Alaaeddin, M. H.; Sapuan, S. M.; Zuhri, M. Y. M.; Zainudin, E. S.; Al-Oqla, Faris M.IOP Conference Series: Materials Science and Engineering (2018), 409 (3rd International Conference on Manufacturing, Material and Metallurgical Engineering, 2018), 012021/1-012021/7CODEN: ICSMGW; ISSN:1757-899X. (IOP Publishing Ltd.)This work features the properties and the common industrial applications of two thermoplastic polymers which are the Polyvinyl fluoride (PVF) and the Polyvinylidene fluoride (PVDF), the polymn. and the manufg. processes of their monomers have been emphasized in this work. It is believed that these two materials are having a no. of shared properties and applications. Their distinguishing characteristics allow them to be utilized in various applications with immense interest to the industrial world. They excel, converge, and slightly diverge in most of their properties with diverse transition phases as well as piezoelec. and pyroelec. effects. Their eminent properties qualify them to be used in a no. of industrial and outdoor applications e.g. insulations, sensing materials, laminations, encapsulations, coatings, membranes, biomaterials, aircraft interiors and photovoltaic applications. In conclusion, this work recommends further in-depth anal. to investigate the correlations between these two polymers and to provide oriented numerical information on their performance.176Report on Carcinogens Background Document for Vinyl Fluoride; Technology Planning and Management Corporation: Durham, NC, 2000. https://ntp.niehs.nih.gov/ntp/newhomeroc/roc10/vf_no_appendices_508.pdf (accessed 2019-11-02).177Kennedy, G. L., Jr. Toxicology of fluorine-containing monomers. Crit. Rev. Toxicol. 1990, 21, 149– 170, DOI: 10.3109/10408449009089877[Crossref], [PubMed], [CAS], Google Scholar177https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXksVemur4%253D&md5=944bfb73a39d10f1119fd04673b924d3Toxicology of fluorine-containing monomersKennedy, G. L., Jr.Critical Reviews in Toxicology (1990), 21 (2), 149-70CODEN: CRTXB2; ISSN:0045-6446.A review with 144 refs. on the toxicol. of bromotrifluroethylene, chlorotrifluoroethylene, hexafluoroacetone, hexafluoroisobutylene, hexafluoropropylene, perfluorobutylene, tetrafluoroethylene, trichloropropene, vinyl fluoride and vinylidine fluoride.178Vinyl Halides (Selected). In Report on Carcinogens, 14th ed.; U.S. Department of Health and Human Services National Toxicology Program, 2016. https://ntp.niehs.nih.gov/ntp/roc/content/profiles/vinylhalides.pdf (accessed 2019-11-02).179IARC Monographs on the Evaluation of Carginogenic Risks to Humans. http://publications.iarc.fr/115 (accessed 2019-11-11).180Bolt, H. M.; Bartsch, H.; Barbin, A. Roles of etheno-DNA adducts in tumorigenicity of olefins. CRC Crit. Rev. Toxicol. 1988, 18, 299– 309, DOI: 10.3109/10408448809037469[Crossref], [CAS], Google Scholar180https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXit1ag&md5=9621c5df595f30a3540b894809d00317Roles of etheno-DNA adducts in the tumorigenicity of olefinsBolt, Hermann M.Critical Reviews in Toxicology (1988), 18 (4), 299-309CODEN: CRTXB2; ISSN:0045-6446.A review with 61 refs. on conditions under which etheno-DNA adducts are formed in vivo and their possible biol. activities.181Ballering, L. A.; Nivard, M. J.; Vogel, E. W. Characterization by two-endpoint comparisons of the genetic toxicity profiles of vinyl chloride and related etheno-adduct forming carcinogens in Drosophila. Carcinogenesis 1996, 17, 1083– 1092, DOI: 10.1093/carcin/17.5.1083[Crossref], [PubMed], [CAS], Google Scholar181https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xjt1KltLY%253D&md5=022ee23845bd6afd02280d13c5042872Characterization by two-endpoint comparisons of the genetic toxicity profiles of vinyl chloride and related etheno-adduct forming carcinogens in DrosophilaBallering, L. A. P.; Nivard, M. J. M.; Vogel, E. W.Carcinogenesis (1996), 17 (5), 1083-1092CODEN: CRNGDP; ISSN:0143-3334. (Oxford University Press)The genetic toxicity profiles of vinyl chloride (VCl), vinyl bromide (VBr), Et carbamate (EC), vinyl carbamate (VC) and some structurally related chems. were investigated in both somatic and germ cells of Drosophila melanogaster. In the white/white+ eye mosaic assay, a screening system measuring predominantly homologous recombination in somatic cells, only marginal genotoxic activities were obsd. for acetyl chloride (ACl), glycol aldehyde (GCA), 2,2'-dichlorodiethyl ether (DDE) and Me carbamate (MC), whereas VCl. 2-Chloroacetaldehyde (CAA), VBr, 2-bromoacetaldehyde (BAA) and EC were clearly recombinogenic in the assay. Those chems. proven to be recombinogenic in somatic cells were investigated further in postmeiotic male germ cells, utilizing as descriptors of their genotoxicity ICL/RL and Mexr-/Mexr+ indexes. The ICL/RL index is the rate of induced chromosome loss (CL), a clastogenic event, dividend by the forward mutation rate, measured as recessive lethal (RL) mutations in 700 loci of the X-chromosome. The Mexr-/Mexr+ mutation enhancement ratio is obtained by detg. RL under excision repair deficient vs. repair proficient conditions. With ICL/RL values (2.7-6.9) similar to those obtained for crosslinking agents, vinyl chloride, vinyl bromide, Et carbamate and vinyl carbamate are all efficient clastogenic agents in Drosophila germ cells. In the absence of excision repair, however, neither CEO nor CAA gave a hyper-mutability response (Mexr-/Mexr+ ≈1). By contrast, VCl, VBr, EC and VC showed clearly enhanced Mexr-/Mexr+ ratios, suggesting that these compds. produce some repairable DNA modification(s) that are not generated by their epoxides. This unexpected finding points to the formation of other, yet unknown, metabolites of vinyl chloride, vinyl bromide, Et carbamate and vinyl carbamate. Our results support the concept that the epoxides chloroethylene oxide (CEO), bromoethylene oxide (BEO) and vinyl carbamate epoxide (VCO) are the most essential mutagenic intermediates. Compared to chloroethylene oxide (CEO), 2-chloroacetaldehyde (CAA) was approx. 50 times less effective in the induction of RL, whereas BAA was inactive as a mutagen. These findings are consistent with the general view that CAA and BAA play no major role in the genotoxic action of vinyl halides.182Cantoreggi, S.; Keller, D. A. Pharmacokinetics and metabolism of vinyl fluoride in vivo and in vitro. Toxicol. Appl. Pharmacol. 1997, 143, 130– 139, DOI: 10.1006/taap.1996.8041[Crossref], [PubMed], [CAS], Google Scholar182https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXhvVSjsLs%253D&md5=7832d84167caf91f3187e0443a046f6dPharmacokinetics and metabolism of vinyl fluoride in vivo and in vitroCantoreggi, Sergio; Keller, Douglas A.Toxicology and Applied Pharmacology (1997), 143 (1), 130-139CODEN: TXAPA9; ISSN:0041-008X. (Academic)In a first set of expts., groups of three rats or five mice were exposed to vinyl fluoride (VF) in a closed-chamber gas uptake system at starting concns. ranging from 50 to 250 ppm. Chamber concns. of VF were measured every 10-12 min by gas chromatog. Partition coeffs. were detd. by the vial equilibration technique and used as parameters for a physiol. based pharmacokinetic (PBPK) model. Mice showed a higher whole-body metabolic capacity compared to rats (Vmaxc = 0.3 vs. 0.1 mg/h-kg). Both species had an estd. Km of ≤0.02 mg/L. The specificity for the oxidn. of VF in vivo was detd. by selective inhibition or induction of CYP 2E1. Inhibition with 4-methylpyrazole completely impaired VF uptake in rats and mice, whereas induction with ethanol (rats only) increased the metabolic capacity by two- to threefold. The pharmacokinetics of VF were also investigated in vitro. Microsomes from rat and mouse liver were incubated in a sealed vial with VF and an NADPH-regenerating system. Headspace concns. (10-300 ppm) were monitored over time by gas chromatog. Consistent with the in vivo data, VF was metabolized faster by mouse microsomes than by rat microsomes (Vmax = 3.5 and 1.1 nmol/h-mg protein, resp.). The rates of metab. by human liver microsomes were generally in the same range as those found with rat liver microsomes (Vmax = 0.5-1.3 nmol/h-mg protein), but one sample was similar to mice (Vmax = 3.3 nmol/h-mg protein). Metabolic rates in human microsomes were found to correlate with the amt. of CYP 2E1 as detd. by Western blotting and by chlorzoxazone 6-hydroxylation. It is concluded that the greater metabolic capacity of mice for VF both in vivo and in vitro may contribute to their greater susceptibility to tumor formation. CYP 2E1 is clearly the main isoenzyme involved in the oxidn. of VF in all species tested. VF pharmacokinetics and metab. in humans may depend upon the interindividual variability in the expression level of CYP 2E1. The excellent correspondence between in vivo and in vitro kinetics in rodents improves substantially the degree of confidence for human in vivo predictions from in vitro data.183Conolly, R. B.; Jaeger, R. J. Acute hepatotoxicity of ethylene and halogenated ethylenes after PCB pretreatment. Environ. Health Perspect. 1977, 21, 131– 135, DOI: 10.1289/ehp.7721131[Crossref], [PubMed], [CAS], Google Scholar183https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXktlOms74%253D&md5=0d3a5c6253e51f2e3bc50928754d8d24Acute hepatotoxicity of ethylene and halogenated ethylenes after PCB pretreatmentConolly, Rory B.; Jaeger, Rudolph J.Environmental Health Perspectives (1977), 21 (), 131-5CODEN: EVHPAZ; ISSN:0091-6765.In rats pretreated with Aroclor 1254 [11097-69-1] (300 μmol/kg, by gavage) inhalation of vinyl chloride [75-01-4] (30,000 ppm) or ethylene [74-85-1] (20,000 ppm), for 4 h, increased serum alanine-α-ketoglutarate transaminase (SAKT) and sorbitol dehydrogenase, resp. within the 8th and the 24th h after exposure was obsd. Exposure temps. of 12.1-30.3° did not affect the hepatotoxicity of the gases; however, at 33.8° both the elevation of SAKT and the mortality were markedly increased. Overnight fasting prior to treatment with vinyl chloride or ethylene increased hepatotoxicity as evidenced by markedly increased sorbitol dehydrogenase. In fasted rats pretreated with Aroclor 1254 and given trichloropropane epoxide (TE) [3083-23-6] a synergistic increase in the toxicity of ethylene and vinyl chloride was obsd. Pretreatment with TE and fasting increased the toxicity of vinyl bromide [593-60-2] while TE, but not fasting, enhanced vinyl fluoride [75-02-5] toxicity. The acute toxicity of the tested compds. may be mediated through epoxide intermediates.184Bolt, H. M.; Peter, H.; Fost, U. Analysis of macromolecular ethylene oxide adducts. Int. Arch. Occup. Environ. Health 1988, 60, 141– 144, DOI: 10.1007/BF00378688[Crossref], [PubMed], [CAS], Google Scholar184https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXlt1ers7k%253D&md5=33d39e8f51888b3f274a70b494cc9b3eAnalysis of macromolecular ethylene oxide adductsBolt, Hermann M.; Peter, Hans; Foest, UlrichInternational Archives of Occupational and Environmental Health (1988), 60 (3), 141-4CODEN: IAEHDW; ISSN:0340-0131.A review with 26 refs. on the formation of macromol. adducts between ethylene oxide and proteins and nucleic acids and methods for their detection.185La, D. K.; Swenberg, J. A. DNA adducts: biological markers of exposure and potential applications to risk assessment. Mutat. Res., Rev. Genet. Toxicol. 1996, 365, 129– 146, DOI: 10.1016/S0165-1110(96)90017-2[Crossref], [PubMed], [CAS], Google Scholar185https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XlvVeitbo%253D&md5=c7acf596e5bf3bc4fc2437f85b2f5f39DNA adducts: biological markers of exposure and potential applications to risk assessmentLa, David K.; Swenberg, James A.Mutation Research, Reviews in Genetic Toxicology (1996), 365 (1-3), 129-146CODEN: MRRTEP; ISSN:0165-1110. (Elsevier B.V.)A review and discussion with many refs. Specificity of DNA adduct formation, relevance of DNA adducts, mutational spectra, DNA repair, current methodologies to measure DNA adducts, quant. relationships between exposure and DNA adducts, potential applications of mol. dosimetry for risk assessment, interspecies extrapolation, etc., are discussed.186Guengerich, F. P. Mechanisms of formation of DNA adducts from ethylene dihalides, vinyl halides, and arylamines. Drug Metab. Rev. 1994, 26, 47– 66, DOI: 10.3109/03602539409029784[Crossref], [PubMed], [CAS], Google Scholar186https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXkslGgtbg%253D&md5=8acb1ad53181eb95905adb52d4bcfaceMechanisms of formation of DNA adducts from ethylene dihalides, vinyl halides, and arylaminesGuengerich, F. PeterDrug Metabolism Reviews (1994), 26 (1-2), 47-66CODEN: DMTRAR; ISSN:0360-2532.A review with 61 refs. This article reviews some studies done in the author's lab. on 3 important classes of carcinogens: ethylene dihalides, vinyl halides, and arylamines.187Swenberg, J. A.; La, D. K.; Scheller, N. A.; Wu, K. Y. Dose-response relationships for carcinogens. Toxicol. Lett. 1995, 82–83, 751– 756, DOI: 10.1016/0378-4274(95)03593-1[Crossref], [PubMed], [CAS], Google Scholar187https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xht1Wrur0%253D&md5=ca3bbcd3d065f7d1fa8a2e2c6a27c565Dose-response relationships for carcinogensSwenberg, James A.; La, David K.; Scheller, Nova A.; Wu, Kuen-yuhToxicology Letters (1995), 82/83 (1-6), 751-6CODEN: TOLED5; ISSN:0378-4274. (Elsevier)A review and discussion with 15 refs. Biotransformation of chem. carcinogens involves both metabolic activation and detoxication. The mol. dose present on DNA as adducts represents a balance between these 2 pathways (formation) and DNA repair. All of these are enzymic processes subject to satn. When none of the pathways is satd., linear mol. dosimetry is expected whereas if metabolic activation is satd., a supralinear response occurs. If detoxication or DNA repair is satd., a sublinear response occurs. With chronic exposure, steady-state concns. of DNA adducts develop and these follow the same patterns. With several alkylating agents, multiple adducts are formed. The extent of formation is chem. defined, but different DNA repair pathways can be involved for different adducts. By understanding the mol. dose and biol. of each adduct and comparing these to the dose-response for tumor induction, it may be possible to identify the most appropriate biomarkers for risk assessment. Recently, endogenous DNA adducts identical to those induced by known human carcinogens have been identified. These endogenously formed adducts may play an important role in human carcinogenesis.188Swenberg, J. A.; Bogdanffy, M. S.; Ham, A.; Holt, S.; Kim, A.; Morinello, E. J.; Ranasinghe, A.; Scheller, N.; Upton, P. B. Formation and repair of DNA adducts in vinyl chloride- and vinyl fluoride-induced carcinogenesis. IARC Sci. Publ. 1999, 29– 43[PubMed], [CAS], Google Scholar188https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXitF2qsbg%253D&md5=37d154fe244e303130c8d0da484d73c5Formation and repair of DNA adducts in vinyl chloride- and vinyl fluoride-induced carcinogenesisSwenberg, J. A.; Bogdanffy, M. S.; Ham, A.; Holt, S.; Kim, A.; Morinello, E. J.; Ranasinghe, A.; Scheller, N.; Upton, P. B.IARC Scientific Publications (1999), 150 (Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis), 29-43CODEN: IARCCD; ISSN:0300-5038. (International Agency for Research on Cancer)A review and discussion with many refs. on the formation and repair of DNA adducts assocd. with vinyl chloride and vinyl fluoride in exposed and control rodents and unexposed humans. Vinyl chloride is a known human and animal carcinogen that induces angiosarcomas of the liver. These vinyl halides induce etheno (ε) adducts that are identical to those formed after lipid peroxidn. Of these adducts, N2,3-ethenoguanine (εG) is present in the greatest amts. in tissues of exposed animals. After exposure to vinyl chloride for 4 wk, εG levels attain steady-state concns., such that the amt. of newly formed adducts equals the no. of adducts that are lost each day. The authors report the first dosimetry of εG in rats exposed to 0, 10, 100, or 1100 ppm vinyl chloride for 5 days or 4 wk. The no. of adducts increased in a supralinear manner. Exposure to 10 ppm vinyl chloride for 5 days caused a 2- to 3-fold increase in εG over that of the controls while a 4-wk exposure resulted in a 5-fold increase. This was confirmed with [13C2]vinyl chloride and by measuring exogenous and endogenous adducts in the same animals. Exposure to 100 ppm vinyl chloride for 4 wk caused a 25-fold increase in εG levels over that found in control rats while exposure to 1100 ppm resulted in a 42-fold increase. The amt. of endogenous εG was similar in liver DNA from rats and humans. A comparable response to exposure was seen in rats and mice exposed to 0, 25, 250, or 2500 ppm vinyl fluoride for 12 mo. There was a very high correlation between εG levels in rat and mouse liver at 12 mo and the incidence of hemangiosarcoma at 2 yr. The authors were able to demonstrate that the target cell population for angiosarcoma, the nonparenchymal cells, contained more εG than hepatocytes, even though nonparenchymal cells are exposed by diffusion of vinyl halide metabolites formed in hepatocytes. The expression of N-methylpurine-DNA glycosylase mRNA was induced in rat liver after exposure to either 25 or 2500 ppm vinyl fluoride. When this induction was investigated in hepatocytes and nonparenchymal cells, it was found that the latter had only 20% of the N-methylpurine-DNA glycosylase mRNA of hepatocytes, and that only the hepatocytes had induction of this expression after exposure to vinyl fluoride. Thus, the target cells for vinyl halide carcinogenesis have much lower expression of this DNA repair enzyme, which has been assocd. with etheno adduct repair.189Filser, J. G.; Jung, P.; Bolt, H. M. Increased acetone exhalation induced by metabolites of halogenated C1 and C2 compounds. Arch. Toxicol. 1982, 49, 107– 116, DOI: 10.1007/BF00332358[Crossref], [PubMed], [CAS], Google Scholar189https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XpvFSisA%253D%253D&md5=49db3c9a9010a263e6ead49675b5887fIncreased acetone exhalation induced by metabolites of halogenated C1 and C2 compoundsFilser, J. G.; Jung, P.; Bolt, H. M.Archives of Toxicology (1982), 49 (2), 107-16CODEN: ARTODN; ISSN:0340-5761.Rats were exposed, in a closed desiccator jar chamber, concns. of various halogenated C1 and C2 compds. at which the metabolizing capacities were satd. (Vmax conditions). Within the exposure period of 50 h concns. of the xenobiotic and of exhaled Me2CO [67-64-1] were monitored in the gas phase of the system. The quant. extent of Me2CO exhalation was dependent on the individual compd. examd. Me2CO exhalation was stimulated in presence of vinyl chloride [75-01-4], vinyl bromide [593-60-2], vinyl fluoride [75-02-5], vinylidene fluoride [75-38-7], cis- [156-59-2] and trans-1,2-dichloroethylene [156-60-5], trichloroethylene [79-01-6], perchloroethylene [127-18-4], CH2Cl2 [75-09-2], CHCl3 [67-66-3], CCl4 [56-23-5], and 1,1,2-trichloroethane [79-00-5]. No stimulation of Me2CO exhalation occurred with 1,1,1-trichloroethane [71-55-6] and with the ref. hydrocarbon n-hexane. Also, Me2CO exhalation was evoked by infusions of either fluoroacetate [144-49-0] or chloroacetate [14526-03-5], 2 anticipated or proven metabolites of some haloethylenes; the infusion rate of which were based on calcns. of the metabolic rates of vinylidene fluoride and of vinyl chloride, resp.190Ortiz de Montellano, P. R.; Kunze, K. L.; Beilan, H. S.; Wheeler, C. Destruction of cytochrome P-450 by vinyl fluoride, fluroxene, and acetylene. Evidence for a radical intermediate in olefin oxidation. Biochemistry 1982, 21, 1331– 1339, DOI: 10.1021/bi00535a035[ACS Full Text], [CAS], Google Scholar190https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XpvFGgtw%253D%253D&md5=c6623d20a258545ddd72e80cdde1750dDestruction of cytochrome P-450 by vinyl fluoride, fluroxene, and acetylene. Evidence for a radical intermediate in olefin oxidationOrtiz de Montellano, Paul R.; Kunze, Kent L.; Beilan, Hal S.; Wheeler, ConradBiochemistry (1982), 21 (6), 1331-9CODEN: BICHAW; ISSN:0006-2960.Vinyl fluoride, vinyl bromide, fluroxene (2,2,2-trifluoroethyl vinyl ether), and acetylene alkylate the prosthetic heme group of cytochrome P-450 enzymes which catalyze their metab. The alkylated heme moiety has been identified in all 4 cases, after carboxyl group methylation and demetalation, as the di-Me ester of N-(2-oxoethyl)protoporphyrin IX. The di-Me acetal deriv. of the aldehyde group in this structure was also isolated. The formation of the same prosthetic heme adduct with the 4 substrates requires introduction of an O at the trifluoroethoxy or halide-substituted terminus of the π bond and reaction of the unsubstituted terminus with a heme N atom. This reaction orientation is consistent with a radical intermediate, possibly formed by way of an initial π-bond radical cation, but is difficult to reconcile with a cationic intermediate. The occurrence of a radical intermediate in the oxidn. of olefins by cytochrome P-450 is thus suggested.191Odum, J.; Green, T. The metabolism and nephrotoxicity of tetrafluoroethylene in the rat. Toxicol. Appl. Pharmacol. 1984, 76, 306– 318, DOI: 10.1016/0041-008X(84)90012-7[Crossref], [PubMed], [CAS], Google Scholar191https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXptV2htw%253D%253D&md5=3d9f3ef28e97c4933a69a0bcaa5cc530The metabolism and nephrotoxicity of tetrafluoroethylene in the ratOdum, J.; Green, T.Toxicology and Applied Pharmacology (1984), 76 (2), 306-18CODEN: TXAPA9; ISSN:0041-008X.Exposure of rats to 6000 ppm tetrafluoroethylene (I) [116-14-3] for 6 h produced marked damage to the proximal tubule of the kidney with no effect on the liver. The toxicity was characterized by very high concns. of urinary glucose [50-99-7] and by marked increases in the concns. of several urinary enzymes. The no obsd. effect level for a 6-h exposure was 2000 ppm. I was metabolized to S-(1,1,2,2-tetrafluoroethyl)glutathione [94840-67-2] by rat liver fractions in vitro; the reaction was catalyzed by microsomal and cytosolic GSH S-transferase [50812-37-8]. The rate with microsomes was 4 times with cytosol fractions. Evidence for this metabolic pathway in vivo was obtained by the identification of the cysteinylglycine and cysteine conjugates of I in rat bile. Cytochrome P 450 [9035-51-2] oxidn., a common metabolic route for haloalkenes, does not appear to occur in the metab. of I. When administered orally to rats, the synthetic cysteine conjugate of I caused renal damage identical to that caused by I itself. The conjugate was metabolized by renal slices in vitro giving pyruvate, NH3, and a reactive species which caused marked inhibition of org. ion transport into slices. Purified renal βb-lyase [68652-57-3] also cleaved this conjugate giving stoichiometric amts. of pyruvate and ammonia. The nephrotoxicity of I may derive from the hepatic GSH conjugate of this compd. Following excretion and degrdn. of this conjugate in bile, the cysteine conjugate is reabsorbed and further metabolized in the kidney by the enzyme β-lyase to a cytotoxic species.192Keller, D. A.; Kennedy, G. L., Jr.; Ross, P. E.; Kelly, D. P.; Elliott, G. S. Toxicity of tetrafluoroethylene and S-(1,1,2, 2-tetrafluoroethyl)-l-cysteine in rats and mice. Toxicol. Sci. 2000, 56, 414– 423, DOI: 10.1093/toxsci/56.2.414[Crossref], [PubMed], [CAS], Google Scholar192https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXlsVCjur0%253D&md5=7d73e93c7116bae45ff2f939915cdaa5Toxicity of tetrafluoroethylene and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine in rats and miceKeller, Douglas A.; Kennedy, Gerald L., Jr.; Ross, Paul E.; Kelly, David P.; Elliott, Glenn S.Toxicological Sciences (2000), 56 (2), 414-423CODEN: TOSCF2; ISSN:1096-6080. (Oxford University Press)Groups of 25 female F344 rats and 25 female B6C3F1 mice were exposed to 0, 30, 300, 600, or 1200 ppm tetrafluoroethylene (TFE) by inhalation for up to 12 days. Another set of 25 female rats and 25 female mice of the same strains were given 0, 5, 20, or 50 mg/kg S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFE-CYS) by oral gavage for 12 days. Both 12-day exposure regimens consisted of exposures for 5 consecutive days, a weekend with no exposures, and 4 consecutive daily exposures following the weekend. Five animals per group were sacrificed after the first exposure, the fifth exposure, and the ninth exposure for evaluation of cell proliferation in the liver and kidney. The remaining animals in each group (up to 10) were sacrificed after the ninth exposure (test day 12) for pathol. evaluation of the liver, kidney, and spleen. Clin. pathol. evaluations were performed on test day 11 or 12. Inhalation of TFE by rats and mice caused slight microscopic changes in the kidneys of rats and mice but no histopathol. changes in the liver. In the kidney, administration of TFE-CYS by gavage caused severe microscopic changes in rats, moderate-to-severe changes in mice, and no microscopic changes in the liver. Cell proliferation was increased in the kidneys of rats and mice given TFE by inhalation and TFE-CYS by gavage. TFE-CYS also caused increased liver wts. and cell proliferation in the liver of rats and mice at the high doses. The cell proliferation response in the kidney and liver was transient in both species, being most pronounced after 5 days of exposure, and less evident or absent after 12 days of exposure. In the kidney, the cell proliferation and histopathol. response in rats was generally more pronounced than in mice. Kidney damage and cell proliferation were confined to the pars recta (P3) of the outer stripe of the outer medulla and medullary rays. Tubules in mice exposed to TFE and TFE-CYS had mostly regenerating cells by test day 12 while in rats the tubules still showed marked degeneration along with regeneration by the end of the study. The cortical labyrinth (P1 and P2 segments) was also affected at the 50-mg/kg dose of TFE-CYS in rats. Rats exposed to 50 mg/kg TFE-CYS had a mild anemia, and rats exposed to 1200 ppm TFE had slight, biol. inconsequential decreases in erythrocyte mass that may have been compd.-related. In spite of the rather pronounced histopathol. changes in the kidneys of rats exposed to TFE-CYS, there was no clin. chem. evidence for decreased kidney function. Increased levels of urinary fluoride were present in rats exposed to 300 ppm and greater of TFE and in rats exposed to 20 and 50 mg/kg TFE-CYS. The spleen was not affected in this study. Overall, the results of this study suggest that the effects of TFE could be attributed to the toxicity of TFE-CYS over the course of a 2-wk exposure, as all effects that were seen with TFE were also seen with TFE-CYS.193Puts, G. J.; Crouse, P.; Ameduri, B. M. Polytetrafluoroethylene: Synthesis and Characterization of the Original Extreme Polymer. Chem. Rev. 2019, 119, 1763– 1805, DOI: 10.1021/acs.chemrev.8b00458[ACS Full Text], [CAS], Google Scholar193https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVCltr0%253D&md5=5bb35db39b39c6c671a2e47c98627689Polytetrafluoroethylene: Synthesis and Characterization of the Original Extreme PolymerPuts, Gerard J.; Crouse, Philip; Ameduri, Bruno M.Chemical Reviews (Washington, DC, United States) (2019), 119 (3), 1763-1805CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. This review aims to be a comprehensive, authoritative, and crit. review of general interest to the chem. community (both academia and industry) as it contains an extensive overview of all published data on the homopolymn. of tetrafluoroethylene (TFE), detailing the TFE homopolymn. process and the resulting chem. and phys. properties. Several reviews and encyclopedia chapters on the properties and applications of fluoropolymers in general were published, including various reviews that extensively report copolymers of TFE (listed below). Despite this, a thorough review of the specific methods of synthesis of the homopolymer, and the relationships between synthesis conditions and the physicochem. properties of the material prepd., has not been available. This review intends to fill that gap. As known, PTFE and its marginally modified derivs. comprise some 60-65% of the total international fluoropolymer market with a global increase of ca. 7% per annum of its prodn. Numerous companies, such as Asahi Glass, Solvay Specialty Polymers, Daikin, DuPont/Chemours, Juhua, 3F and 3M/Dyneon, etc., produce TFE homopolymers. Such polymers, both high mol.-mass materials and waxes, are chem. inert, hydrophobic, and exhibit an excellent thermal stability and an exceptionally low coeff. of friction. These polymers find use in applications ranging from coatings and lubrication to pyrotechnics, and an extensive industry (electronic, aerospace, wires and cables, and textiles) was built around them. South Africa, being the third largest producer of fluorspar (CaF2), the precursor to hydrogen fluoride and fluorine, has embarked on an industrial initiative to locally beneficiate its fluorspar reserves, with the local prodn. of fluoropolymers being one projected outcome. As our manuscript focuses specifically on the homopolymn. of TFE (the starting point for all fluoropolymer industries), it will be of considerable use to start-up companies and other com. entities looking to enter the fluoropolymer market, and end-user companies. The manuscript commences with a short discussion on the synthesis and prodn. of TFE (both at industrial and lab. scales), including the safety aspects surrounding handling (since that monomer is regarded as explosive if brought into contact with oxygen due to the formation of peroxides), transport and storage, and then expands into detailed discussions dealing with aspects such as the various additives used (buffers, chain transfer agents, surfactants etc.), the solvent environment, and the reaction conditions. A further section reports the properties of PTFE with respect to the polymn. conditions and an overview on the specialized techniques used to characterize PTFE. Finally, the applications of PTFE into various topics, ranging from elec. insulation, tribol. to medical applications, and chem. resistant coatings and pyrotechnics are discussed.194NTP Technical Report on the Toxicology and Carcinogenesis Studies of Tetrafluoroethylene (CAS No. 116-14-3) in F344/N Rats and B6C3F1 Mice (Inhalation Studies). National Institutes of Health National Toxicology Program, April 1997. https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr450.pdf (accessed 2019-11-02).195Shim, J. Y.; Richard, A. M. Theoretical evaluation of two plausible routes for bioactivation of S-(1,1-difluoro-2,2-dihaloethyl)-l-cysteine conjugates: thiirane vs thionoacyl fluoride pathway. Chem. Res. Toxicol. 1997, 10, 103– 110, DOI: 10.1021/tx9600863[ACS Full Text], [CAS], Google Scholar195https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXotVWrtg%253D%253D&md5=672ddea96d18a414cc6cb5b869a8000dTheoretical Evaluation of Two Plausible Routes for Bioactivation of S-(1,1-Difluoro-2,2-dihaloethyl)-L-cysteine Conjugates: Thiirane vs. Thionoacyl Fluoride PathwayShim, Joong-Youn; Richard, Ann M.Chemical Research in Toxicology (1997), 10 (1), 103-110CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)Computational methods were applied to the investigation of two proposed metabolic pathways leading from the thiolate to either a thiirane or thionoacyl fluoride intermediate, both electrophilic species presumed capable of binding to proteins or DNA. Studied were six F-, Cl-, and Br-substituted 2,2-dihalo-1,1-difluoroethane-1-thiolates (2,2-dihalo-DFETs). Pathway preference was detd. for each thiolate by comparison of reaction energy profiles and activation energies. At all but the lowest level of ab initio theory, a thionoacyl fluoride pathway was predicted for 2,2-difluoro-DFET, while a thiirane pathway was energetically preferred for the brominated 2,2-dihalo-DFETs. These results offer a clear mechanism-based rationale for distinguishing 2,2-difluoro-DFET from the brominated 2,2-dihalo-DFETs, while the results are less clear for the 2,2-dichloro and 2-chloro-2-fluoro-DFETs, which at the highest level of ab initio treatment had a relatively small energy preference (2.4 kcal/mol) for the thiirane pathway. The predicted clear preference for a thiirane pathway for the brominated 2,2-dihalo-DFETs is not consistent with a recently proposed pathway involving α-thiolactone formation through a thionoacyl fluoride intermediate [Finkelstein, M. B., et al. (1995) J. Am. Chem. Soc. 117, 9590-9591], but is supported by results of a recent study providing exptl. evidence for thiirane formation from the brominated 2,2-dihalo-DFETs [Finkelstein, M. B., et al. (1996) Chem. Res. Toxicol. 9, 227-231].196Commandeur, J. N.; Brakenhoff, J. P.; De Kanter, F. J.; Vermeulen, N. P. Nephrotoxicity of mercapturic acids of three structurally related 2,2-difluoroethylenes in the rat. Indications for different bioactivation mechanisms. Biochem. Pharmacol. 1988, 37, 4495– 4504, DOI: 10.1016/0006-2952(88)90665-X[Crossref], [PubMed], [CAS], Google Scholar196https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXptVSltQ%253D%253D&md5=7d98aae03afd57c671238993632ebbd3Nephrotoxicity of mercapturic acids of three structurally related 2,2-difluoroethylenes in the rat. Indications for different bioactivation mechanismsCommandeur, J. N. M.; Brakenhoff, J. P. G.; De Kanter, F. J. J.; Vermeulen, N. P. E.Biochemical Pharmacology (1988), 37 (23), 4495-504CODEN: BCPCA6; ISSN:0006-2952.The biotransformation and the hepato- and nephrotoxicity of the mercapturic acids (N-acetyl-1-cysteine S-conjugates) of three structurally related 2,2-difluoroethylenes were investigated in vivo in the rat. All mercapturic acids appeared to cause nephrotoxicity, without any measurable effect on the liver. The mercapturic acid of tetrafluoroethylene (TFE-NAC) appeared to be the most potent nephrotoxin, causing toxicity upon an i.p. dose of 50 μmol/kg. The mercapturic acids of 1,1-dichloro-2,2-difluoroethylene (DCDFE-NAC) and 1,1-dibromo-2,2-difluoroethylene (DBDFE-NAC) were nephrotoxic at slightly higher doses, i.e. at 75 and 100 μmol/kg, resp. In the urine of TFE-NAC-treated rats significant amts. of difluoroacetic acid (DFAA) could be detected. With increasing doses, the relative amt. of DFAA in urine increased progressively (5-18% of dose). In urine of rats treated with DCDFE-NAC and DBDFE-NAC, however, the corresponding dihaloacetic acids, dichloroacetic acid, and dibromoacetic acid, could not be detected. Formation of DFAA and pyruvate could also be obsd. during in vitro metab. of the cysteine conjugate of tetrafluoroethylene (TFE-CYS) by rat renal cytosol. Inhibition by aminooxyacetic acid pointed to a β-lyase dependency for the DFAA-formation. Next to DFAA and pyruvate, also formation of hydrogen sulfide and thiosulfate could be detected. These results suggest that TFE-CYS is bioactivated to a significant extent to difluorothionacyl fluoride, which most likely is subsequently hydrolyzed to difluorothio(no)acetic acid and difluoroacetic acid. According to formation of pyruvate, the cysteine conjugates derived from DCDFE-NAC and DBDFE-NAC also were efficiently metabolized by rat renal β-lyase. However, the formation of corresponding dihaloacetic acids, dichloroacetic acid, and dibromoacetic acid, could not be detected in vitro at all. Only very small amts. of hydrogen sulfide and thiosulfate were detected. Evidently bioactivation of the latter two conjugates to a dichloro- or dibromothionoacyl fluoride represents only a minor route. Because of better leaving group abilities of chloride and bromide compared to fluoride, rearrangement of the initially formed ethanethiol to a thiirane might be favored. Based on the present in vivo and in vitro data, it is concluded that the nephrotoxicity of the structurally related mercapturic acids of 2,2-difluoroethylenes is dependent on halogen substitution and presumably the result of at least two different mechanisms of bioactivation.197Potter, C. L.; Gandolfi, A. J.; Nagle, R.; Clayton, J. W. Effects of inhaled chlorotrifluoroethylene and hexafluoropropene on the rat kidney. Toxicol. Appl. Pharmacol. 1981, 59, 431– 440, DOI: 10.1016/0041-008X(81)90295-7[Crossref], [PubMed], [CAS], Google Scholar197https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXkvFWqsLw%253D&md5=5a70e9211a2487aa7a174fae8bd1fc1eEffects of inhaled chlorotrifluoroethylene and hexafluoropropene on the rat kidneyPotter, C. L.; Gandolfi, A. J.; Nagle, R.; Clayton, J. W.Toxicology and Applied Pharmacology (1981), 59 (3), 431-40CODEN: TXAPA9; ISSN:0041-008X.Male rats were subjected to a 4.0-h inhalation exposure to chlorotrifluoroethylene (CTFE) [79-38-9] (100-540 ppm) or hexafluoropropene (HFP) [116-15-4] (380-1200 ppm). Within 2 days following exposure, the rats exhibited dose-related proximal tubular necrosis, diuresis, increases in urinary fluoride, urinary lactic dehydrogenase (LDH) [9001-60-9] activity, serum creatinine [60-27-5], and BUN. The toxicities of CTFE and HFP were similar except that CTFE was the more potent renal toxin and HFP produced necrosis of the pars recta and pars convoluta portions of the proximal tubule, while CTFE produced necrosis of only the pars recta. At the lowest exposure concns., diuresis was the most sensitive index of toxicity manifesting 50% increases in water intake and 25% decreases in urine osmolality. Increases in urinary LDH activity correlated with the degree of proximal renal tubular necrosis, with ≥100-fold increases at the highest concns. of CTFE and HFP. At 100 ppm, CTFE induced renal dysfunction (mild diuresis), but no significant increase in urinary LDH nor necrosis was apparent. All concns. of HFP studied produced necrosis within 24 h postexposure with tubular cell regeneration apparent within 4 days.198Commandeur, J. N.; Oostendorp, R. A.; Schoofs, P. R.; Xu, B.; Vermeulen, N. P. Nephrotoxicity and hepatotoxicity of 1,1-dichloro-2,2-difluoroethylene in the rat. Indications for differential mechanisms of bioactivation. Biochem. Pharmacol. 1987, 36, 4229– 4237, DOI: 10.1016/0006-2952(87)90663-0[Crossref], [PubMed], [CAS], Google Scholar198https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXntVaqtg%253D%253D&md5=d01930644460f08f06c66493ef9f4145Nephrotoxicity and hepatotoxicity of 1,1-dichloro-2,2-difluoroethylene in the rat. Indications for differential mechanisms of bioactivationCommandeur, J. N. M.; Oostendorp, R. A. J.; Schoofs, P. R.; Xu, B.; Vermeulen, N. P. E.Biochemical Pharmacology (1987), 36 (24), 4229-37CODEN: BCPCA6; ISSN:0006-2952.1,1-Dichloro-2,2-difluoroethylene (DCDFE) produced marked nephrotoxicity in rats after an i.p. dose of 150 μmol/kg. At doses higher than 375 μmol/kg, DCDFE also produced hepatotoxicity. Aminooxyacetic acid, an inhibitor of cysteine conjugate β-lyase, appeared to be slightly nephrotoxic in rats. Nevertheless it exerted an inhibitory effect on the nephrotoxicity of DCDFE. The N-acetylcysteine conjugate of DCDFE was identified as a major urinary metabolite of DCDFE. When administered as such, this conjugate appeared to be a potent nephrotoxin, without any effect on the liver, indicating that glutathione conjugation of DCDFE is most likely a bioactivation step for nephrotoxicity. The appearance of traces of chlorodifluoroacetic acid in urine of rats treated with higher doses of DCDFE indicates the existence of an oxidative pathway of metab. of DCDFE, probably involving epoxidn. by hepatic mixed-function oxidases. It is speculated that the latter route might account for the hepatotoxicity at higher doses of DCDFE. The nephro- and hepatotoxicity of DCDFE, therefore, most likely are the result of 2 different mechanisms of bioactivation.199Ishmael, J.; Lock, E. A. Nephrotoxicity of hexachlorobutadiene and its glutathione-derived conjugates. Toxicol. Pathol. 1986, 14, 258– 262, DOI: 10.1177/019262338601400216[Crossref], [PubMed], [CAS], Google Scholar199https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXitVyqtrg%253D&md5=5cabde1e399a4d55ab3ba70e1c99bbbaNephrotoxicity of hexachlorobutadiene and its glutathione-derived conjugatesIshmael, John; Lock, Edward A.Toxicologic Pathology (1986), 14 (2), 258-62CODEN: TOPADD; ISSN:0192-6233.The nephrotoxicity of hexachlor-1,3-butadiene (HCBD) [87-68-3], its GSH conjugate (HCBD-GSH) [89021-88-5], cysteine conjugate (HCBD-CYS) [87619-82-7], and its N-acetylcysteine conjugate (HCBD-NAC) [89784-39-4] were compared in male and female Alderley Park rats. Rats, 6-8 wk of age, were given a single i.p. injection of HCBD or its conjugates and killed 24 h later. Nephrotoxicity was assessed by histol. examn. and plasma urea. All 3 conjugates produced an elevation of plasma urea and proximal renal tubular necrosis with a similar localization in the pars recta as seen with HCBD. All the conjugates were more nephrotoxic than HCBD itself. HCBD was ∼4 times more toxic to female rats than to males. This sex difference is also shown by all the HCBD metabolites.200Tysoe, C.; Withers, S. G. Fluorinated mechanism-based inhibitors: common themes and recent developments. Curr. Top. Med. Chem. 2014, 14, 865– 874, DOI: 10.2174/1568026614666140202204602[Crossref], [PubMed], [CAS], Google Scholar200https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXlt1Srsbs%253D&md5=033531feb3cb8aaae337fa4ade61af3bFluorinated Mechanism-Based Inhibitors: Common Themes and Recent DevelopmentsTysoe, Christina; Withers, Stephen G.Current Topics in Medicinal Chemistry (Sharjah, United Arab Emirates) (2014), 14 (7), 865-874CODEN: CTMCCL; ISSN:1568-0266. (Bentham Science Publishers Ltd.)A review. Mechanism-based inhibitors are relatively chem. inert compds. that become activated when processed by their target enzyme, leading to covalent enzyme inactivation. Fluorine substitution confers a no. of properties that are beneficial to the chem. of such inhibitors and to their potential use as pharmaceuticals, and indeed several fluorinated mechanism-based inhibitors have made it to clin. usage over the past 50 years. Well-known examples are the 5- fluorouracil metabolite, 5-fluoro-2'-deoxyuridine-5'-monophosphate, which is used in the treatment of cancer, and α - difluoromethylornithine for the treatment of African sleeping sickness. As the prevalence of fluorine in medicinal chem. continues to rise, more and more medically relevant fluorinated mechanism-based inhibitors are being developed with a variety of interesting properties and uses.201Belter, A.; Skupinska, M.; Giel-Pietraszuk, M.; Grabarkiewicz, T.; Rychlewski, L.; Barciszewski, J. Squalene monooxygenase - a target for hypercholesterolemic therapy. Biol. Chem. 2011, 392, 1053– 1075, DOI: 10.1515/BC.2011.195[Crossref], [PubMed], [CAS], Google Scholar201https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XivFSrtLk%253D&md5=9d4af8d79ed6ed0797c74c6b53a42ddbSqualene monooxygenase - a target for hypercholesterolemic therapyBelter, Agnieszka; Skupinska, Miroslawa; Giel-Pietraszuk, Malgorzata; Grabarkiewicz, Tomasz; Rychlewski, Leszek; Barciszewski, JanBiological Chemistry (2011), 392 (12), 1053-1075CODEN: BICHF3; ISSN:1431-6730. (Walter de Gruyter GmbH & Co. KG)A review. Squalene monooxygenase catalyzes the epoxidn. of C-C double bond of squalene to yield 2,3-oxidosqualene, the key step of sterol biosynthesis pathways in eukaryotes. Sterols are essential compds. of these organisms and squalene epoxidn. is an important regulatory point in their synthesis. Squalene monooxygenase downregulation in vertebrates and fungi decreases synthesis of cholesterol and ergosterol, resp., which makes squalene monooxygenase a potent and attractive target of hypercholesterolemia and antifungal therapies. Currently some fungal squalene monooxygenase inhibitors (terbinafine, naftifine, butenafine) are in clin. use, whereas mammalian enzymes' inhibitors are still under investigation. Research on new squalene monooxygenase inhibitors is important due to the prevalence of hypercholesterolemia and the lack of both sufficient and safe remedies. In this paper we (i) review data on activity and the structure of squalene monooxygenase, (ii) present its inhibitors, (iii) compare current strategies of lowering cholesterol level in blood with some of the most promising strategies, (iv) underline advantages of squalene monooxygenase as a target for hypercholesterolemia therapy, and (v) discuss safety concerns about hypercholesterolemia therapy based on inhibition of cellular cholesterol biosynthesis and potential usage of squalene monooxygenase inhibitors in clin. practice. After many years of use of statins there is some clin. evidence for their adverse effects and only partial effectiveness. Currently they are drugs of choice but are used with many restrictions, esp. in case of children, elderly patients and women of childbearing potential. Certainly, for the next few years, statins will continue to be a suitable tool for cost-effective cardiovascular prevention; however research on new hypolipidemic drugs is highly desirable. We suggest that squalene monooxygenase inhibitors could become the hypocholesterolemic agents of the future.202Cirmena, G.; Franceschelli, P.; Isnaldi, E.; Ferrando, L.; De Mariano, M.; Ballestrero, A.; Zoppoli, G. Squalene epoxidase as a promising metabolic target in cancer treatment. Cancer Lett. 2018, 425, 13– 20, DOI: 10.1016/j.canlet.2018.03.034[Crossref], [PubMed], [CAS], Google Scholar202https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntVOit70%253D&md5=43d5d59d5d9fd3b035826480232ee302Squalene epoxidase as a promising metabolic target in cancer treatmentCirmena, Gabriella; Franceschelli, Paola; Isnaldi, Edoardo; Ferrando, Lorenzo; De Mariano, Marilena; Ballestrero, Alberto; Zoppoli, GabrieleCancer Letters (New York, NY, United States) (2018), 425 (), 13-20CODEN: CALEDQ; ISSN:0304-3835. (Elsevier)A review. Oncogenic alteration of the cholesterol synthesis pathway is a recognized mechanism of metabolic adaptation. In the present review, we focus on squalene epoxidase (SE), one of the two rate-limiting enzymes in cholesterol synthesis, retracing its history since its discovery as an antimycotic target to its description as an emerging metabolic oncogene by amplification with clin. relevance in cancer. We review the published literature assessing the assocn. between SE over-expression and poor prognosis in this disease. We assess the works demonstrating how SE promotes tumor cell proliferation and migration, and displaying evidence of cancer cell demise in presence of human SE inhibitors in in vitro and in vivo models. Taken together, robust scientific evidence has by now accumulated pointing out SE as a promising novel therapeutic target in cancer treatment.203Ono, T.; Nakazono, K.; Kosaka, H. Purification and partial characterization of squalene epoxidase from rat liver microsomes. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1982, 709, 84– 90, DOI: 10.1016/0167-4838(82)90424-1[Crossref], [PubMed], [CAS], Google Scholar203https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3sXhtlGhtg%253D%253D&md5=7ed26117db6e34c54a37e21eebb86e3bPurification and partial characterization of squalene epoxidase from rat liver microsomesOno, Teruo; Nakazono, Kiyoshi; Kosaka, HirokoBiochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (1982), 709 (1), 84-90CODEN: BBAEDZ; ISSN:0167-4838.Squalene epoxidase (EC 1.14.99.7) (I) was purified to apparent homogeneity from rat liver microsomes. The purifn. was carried out by solubilization of microsomes by Triton X-100, fractionation with ion-exchangers, hydroxyapatite, Cibacron Blue Sepharose 4B, and chromatofocusing column chromatog. A total purifn. of 143-fold over the 1st DEAE-cellulose fraction was achieved. Purified I gave a single major band on SDS-polyacrylamide gel electrophoresis and the mol. wt. was estd. to be 51,000 as a single polypeptide chain. I showed no distinct absorption spectrum in the visible regions. The I activity was reconstituted with the purified enzyme, NADPH-cytochrome P 450 reductase, FAD, NADPH, and O2 in the presence of Triton X-100. The apparent Km for squalene and FAD were 13 and 5 μM, resp. The Vmax was ∼186 nmol/mg protein/30 min for 2,3-oxidosqualene. I activity was not inhibited by potent inhibitors of cytochrome P 450. Apparently, I is distinct from cytochrome P 450 isoenzymes.204Moore, W. R.; Schatzman, G. L.; Jarvi, E. T.; Gross, R. S.; McCarthy, J. R. Terminal difluoro olefin analogs of squalene are time-dependent inhibitors of squalene epoxidase. J. Am. Chem. Soc. 1992, 114, 360– 361, DOI: 10.1021/ja00027a056[ACS Full Text], [CAS], Google Scholar204https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xjs1Oqtg%253D%253D&md5=00d486607e27a6cce124e6925eacf244Terminal difluoro olefin analogs of squalene are time-dependent inhibitors of squalene epoxidaseMoore, William R.; Schatzman, Gerald L.; Jarvi, Esa T.; Gross, Raymond S.; McCarthy, James R.Journal of the American Chemical Society (1992), 114 (1), 360-1CODEN: JACSAT; ISSN:0002-7863.Squalene epoxidase is an essential enzyme in the biosynthesis of cholesterol in humans and ergosterol in fungi and thus represents a potential target for hypocholesterolemic and antifungal therapeutics. The design, synthesis, and enzyme inhibition data for a series of mono- and difluoro olefin squalene analogs, such as (E,E,E,E)-1,1-difluoro-5,9,14,18,22-pentamethyl-1,5,9,13,17,21-tricosahexaene (I), are reported. Difluoro olefin squalene analogs I and II were time-dependent inhibitors of squalene epoxidase from rat liver; Ki and kinact values were 4 μM and 0.16 min-1 and 8 μM and 0.12 min-1 for I and II, resp. Difluoro olefin compds. with subtle structural changes from I had substantially reduced potency: a homolog of I increased in chain length and a C-2 Me-substituted I were not time-dependent inhibitors and had IC50 values of >100 μM. The inhibition of squalene epoxidase by difluoro olefins I and II appeared to be enzyme-activated. Monofluoro olefin squalene analogs were reversible inhibitors of squalene epoxidase from rat liver with IC50 values of 100 and 47 μM, resp. A new method for the synthesis of 1-(alkyl)-1 fluoro olefins is presented.205Wolfe, M. S.; Borchardt, R. T. S-adenosyl-l-homocysteine hydrolase as a target for antiviral chemotherapy. J. Med. Chem. 1991, 34, 1521– 1530, DOI: 10.1021/jm00109a001[ACS Full Text], [CAS], Google Scholar205https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXit1Smt70%253D&md5=445e649eb2925474f4b7abade9ab0cb6S-Adenosyl-L-homocysteine hydrolase as a target for antiviral chemotherapyWolfe, Michael S.; Borchardt, Ronald T.Journal of Medicinal Chemistry (1991), 34 (5), 1521-30CODEN: JMCMAR; ISSN:0022-2623.A review with 117 refs. on the antiviral S-adenosylhomocysteine hydrolase inhibitors with discussions on their cytotoxicity and present and future prospects as antiviral drugs as well as tools for studying the physiol. function of the hydrolase.206Reddy, M. C.; Kuppan, G.; Shetty, N. D.; Owen, J. L.; Ioerger, T. R.; Sacchettini, J. C. Crystal structures of Mycobacterium tuberculosis S-adenosyl-l-homocysteine hydrolase in ternary complex with substrate and inhibitors. Protein Sci. 2008, 17, 2134– 2144, DOI: 10.1110/ps.038125.108[Crossref], [PubMed], [CAS], Google Scholar206https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVKgt7vI&md5=c512b956d68fc1db9fcfc2af88fd259aCrystal structures of Mycobacterium tuberculosis S-adenosyl-L-homocysteine hydrolase in ternary complex with substrate and inhibitorsReddy, Manchi C. M.; Kuppan, Gokulan; Shetty, Nishant D.; Owen, Joshua L.; Ioerger, Thomas R.; Sacchettini, James C.Protein Science (2008), 17 (12), 2134-2144CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)S-adenosylhomocysteine hydrolase (SAHH) is a ubiquitous enzyme that plays a central role in methylation-based processes by maintaining the intracellular balance between S-adenosylhomocysteine (SAH) and S-adenosylmethionine. We report the first prokaryotic crystal structure of SAHH, from Mycobacterium tuberculosis (Mtb), in complex with adenosine (ADO) and NAD. Structures of complexes with three inhibitors are also reported: 3'-keto aristeromycin (ARI), 2-fluoroadenosine, and 3-deazaadenosine. The ARI complex is the first reported structure of SAHH complexed with this inhibitor, and confirms the oxidn. of the 3' hydroxyl to a planar keto group, consistent with its prediction as a mechanism-based inhibitor. We demonstrate the in vivo enzyme inhibition activity of the three inhibitors and also show that 2-fluoroadenosine has bactericidal activity. While most of the residues lining the ADO-binding pocket are identical between Mtb and human SAHH, less is known about the binding mode of the homocysteine (HCY) appendage of the full substrate. We report the 2.0 Å resoln. structure of the complex of SAHH cocrystd. with SAH. The most striking change in the structure is that binding of HCY forces a rotation of His-363 around the backbone to flip out of contact with the 5' hydroxyl of the ADO and opens access to a nearby channel that leads to the surface. This complex suggests that His-363 acts as a switch that opens up to permit binding of substrate, then closes down after release of the cleaved HCY. Differences in the entrance to this access channel between human and Mtb SAHH are identified.207Mehdi, S.; Jarvi, E. T.; Koehl, J. R.; McCarthy, J. R.; Bey, P. The mechanism of inhibition of S-adenosyl-l-homocysteine hydrolase by fluorine-containing adenosine analogs. J. Enzyme Inhib. 1990, 4, 1– 13, DOI: 10.3109/14756369009030383[Crossref], [PubMed], [CAS], Google Scholar207https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXhslWhtg%253D%253D&md5=8855633bcc7b81de34f12490b124bcd3The mechanism of inhibition of S-adenosyl-L-homocysteine hydrolase by fluorine-containing adenosine analogsMehdi, Shujaath; Jarvi, Esa T.; Koehl, Jack R.; McCarthy, James R.; Bey, PhilippeJournal of Enzyme Inhibition (1990), 4 (1), 1-13CODEN: ENINEG; ISSN:8755-5093.(Z)-4',5'-Didehydro-5'-deoxy-5'-fluoroadenosine (I), 5'-deoxy-5'-difluoroadenosine (II), and 4',5'-didehydro-5'-deoxy-5'-fluoroarabinosyl-adenosine (III) are inhibitors of rat liver S-adenosyl-L-homocysteine hydrolase. I and II are time-dependent and irreversible inhibitors of the enzyme. Both I and II are oxidized by enzyme-bound NAD to produce NADH, and fluoride anion is formed in the inactivation reaction (0.7 to 1.0 mol fluoride/mol of enzyme subunit, and 1.7 mol fluoride/mol of enzyme subunit from I and II, resp.). The enzyme is stoichiometrically labeled with [8-3H]-I, but the label is lost upon denaturation of the protein either with or without treatment of the labeled complex with sodium borohydride. III, the arabino deriv. of I, is a competitive inhibitor of the enzyme. The mechanism of the inhibition of S-adenosyl-L-homocysteine hydrolase by these inhibitors is discussed.208Lee, K. M.; Choi, W. J.; Lee, Y.; Lee, H. J.; Zhao, L. X.; Lee, H. W.; Park, J. G.; Kim, H. O.; Hwang, K. Y.; Heo, Y. S.; Choi, S.; Jeong, L. S. X-ray crystal structure and binding mode analysis of human S-adenosylhomocysteine hydrolase complexed with novel mechanism-based inhibitors, haloneplanocin A analogues. J. Med. Chem. 2011, 54, 930– 938, DOI: 10.1021/jm1010836[ACS Full Text], [CAS], Google Scholar208https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXlt1Ortw%253D%253D&md5=667394f7f2c22c2aed24f9a4a84bc7a9X-ray Crystal Structure and Binding Mode Analysis of Human S-Adenosylhomocysteine Hydrolase Complexed with Novel Mechanism-Based Inhibitors, Haloneplanocin A AnaloguesLee, Kang Man; Choi, Won Jun; Lee, Yoonji; Lee, Hyun Joo; Zhao, Long Xuan; Lee, Hyuk Woo; Park, Jae Gyu; Kim, Hea Ok; Hwang, Kwang Yeon; Heo, Yong-Seok; Choi, Sun; Jeong, Lak ShinJournal of Medicinal Chemistry (2011), 54 (4), 930-938CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The x-ray crystal structure of human S-adenosylhomocysteine (AdoHcy) hydrolase was first detd. as a tetrameric form bound with the novel mechanism-based inhibitor fluoroneplanocin A (4b). The crystd. enzyme complex showed the closed conformation and turned out to be the intermediate of mechanism-based inhibition. It confirmed that the cofactor depletion by 3'-oxidn. of fluoroneplanocin A contributes to the enzyme inhibition along with the irreversible covalent modification of AdoHcy hydrolase. In addn., a series of haloneplanocin A analogs (4b-e and 5b-e) were designed and synthesized to characterize the binding role and reactivity of the halogen substituents and the 4'-CH2OH group. The biol. evaluation and mol. modeling studies identified the key pharmacophores and structural requirements for the inhibitor binding of AdoHcy hydrolase. The inhibitory activity was decreased as the size of the halogen atom increased and/or if the 4'-CH2OH group was absent. These results could be utilized to design new therapeutic agents operating via AdoHcy hydrolase inhibition.209Jeong, L. S.; Yoo, S. J.; Lee, K. M.; Koo, M. J.; Choi, W. J.; Kim, H. O.; Moon, H. R.; Lee, M. Y.; Park, J. G.; Lee, S. K.; Chun, M. W. Design, synthesis, and biological evaluation of fluoroneplanocin A as the novel mechanism-based inhibitor of S-adenosylhomocysteine hydrolase. J. Med. Chem. 2003, 46, 201– 203, DOI: 10.1021/jm025557z[ACS Full Text], [CAS], Google Scholar209https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XpsVegtbo%253D&md5=1d68e769c44b8cf937fb1ed50aba7acfDesign, Synthesis, and Biological Evaluation of Fluoroneplanocin A as the Novel Mechanism-Based Inhibitor of S-Adenosylhomocysteine HydrolaseJeong, Lak Shin; Yoo, Su Jeong; Lee, Kang Man; Koo, Mi Jeong; Choi, Won Jun; Kim, Hea Ok; Moon, Hyung Ryong; Lee, Min Young; Park, Jae Gyu; Lee, Sang Kook; Chun, Moon WooJournal of Medicinal Chemistry (2003), 46 (2), 201-203CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Fluoroneplanocin A was designed as a novel mechanism-based inhibitor of S-adenosylhomocysteine hydrolase (SAH) and efficiently synthesized via an electrophilic vinyl fluorination reaction (n-BuLi, N-fluorobenzenesulfonimide at -78 °C). Fluoroneplanocin A exhibited 2-fold more potent SAH inhibitory activity than the parent neplanocin A. A new mechanism of irreversible inhibition discovered in this work might provide new alternatives in the design of a different class of antiviral agents operating via SAH inhibition.210Rosen, T. C.; Yoshida, S.; Frohlich, R.; Kirk, K. L.; Haufe, G. Fluorinated phenylcyclopropylamines. 2. Effects of aromatic ring substitution and of absolute configuration on inhibition of microbial tyramine oxidase. J. Med. Chem. 2004, 47, 5860– 5871, DOI: 10.1021/jm049957t[ACS Full Text], [CAS], Google Scholar210https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXoslSgtr4%253D&md5=f997059cce8757712c33518b96a56677Fluorinated Phenylcyclopropylamines. 2. Effects of Aromatic Ring Substitution and of Absolute Configuration on Inhibition of Microbial Tyramine OxidaseRosen, Thomas C.; Yoshida, Shinichi; Froehlich, Roland; Kirk, Kenneth L.; Haufe, GuenterJournal of Medicinal Chemistry (2004), 47 (24), 5860-5871CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of para-substituted diastereopure cis- and trans-2-fluoro-2-arylcyclopropylamines were synthesized and these were investigated as inhibitors of microbial tyramine oxidase from Arthrobacter sp. All compds. were shown to be competitive inhibitors of this enzyme. The nature of the para-substituents in the more potent trans-isomer (cis-relationship between fluorine and the amino group) of 2-fluoro-2-arylcyclopropylamine influenced the inhibitory potency in a consistent fashion. Thus, electron-withdrawing groups (F, Cl) slightly decreased the activity, while the Me group (+ I substituent) increased the activity by a factor of ∼ 7 compared to trans-2-fluoro-2-phenylcyclopropylamine and by a factor of 90 compared to tranylcypromine. Activity also was strongly dependent on the abs. configuration. The (1S,2S)-enantiomer of 2-fluoro-2-phenylcyclopropylamine was an excellent inhibitor of tyramine oxidase whereas the (1R,2R)-enantiomer was essentially devoid of activity.211McDonald, I. A.; Lacoste, J. M.; Bey, P.; Palfreyman, M. G.; Zreika, M. Enzyme-activated irreversible inhibitors of monoamine oxidase: phenylallylamine structure-activity relationships. J. Med. Chem. 1985, 28, 186– 193, DOI: 10.1021/jm00380a007[ACS Full Text], [CAS], Google Scholar211https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXlvValsg%253D%253D&md5=c7f4e34858042219fd1395e6e144a086Enzyme-activated irreversible inhibitors of monoamine oxidase: phenylallylamine structure-activity relationshipsMcDonald, Ian A.; Lacoste, Jean Michel; Bey, Philippe; Palfreyman, Michael G.; Zreika, MoniqueJournal of Medicinal Chemistry (1985), 28 (2), 186-93CODEN: JMCMAR; ISSN:0022-2623.The title compds. I (R and R1 = H, Br, Cl or F; R2 = H or OMe; R3 = H, OH, OMe or CF3; R4 = H, Cl, OH, Me, or OMe) were prepd. as HCl salts by several routes and evaluated in vitro by incubation with a crude rat brain mitochondrial monoamine oxidase (MAO) prepn., and in vivo in mice. With the exception of (Z)-3-chloro-2-phenylallylamine, which acted as a competitive inhibitor, all I caused a time-dependent inhibition of MAO when selective substrates were used. (E)-3-Fluoro-2-(4-methoxyphenyl)- and 2-(3,4-dimethoxyphenyl)-3-fluoroallylamine were as selective for the B form of MAO as deprenyl. (E)-3-Fluoro-2-phenylallylamine given to mice at 1 mg/kg, i.p., gave good inhibition of MAO in the brain and heart which lasted ≤48 h. Structure-activity relations were discussed.212Bortolato, M.; Chen, K.; Shih, J. C. Monoamine oxidase inactivation: from pathophysiology to therapeutics. Adv. Drug Delivery Rev. 2008, 60, 1527– 1533, DOI: 10.1016/j.addr.2008.06.002[Crossref], [PubMed], [CAS], Google Scholar212https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtFWltbbK&md5=9085ccfdebab364e9ea78995bcfc16d1Monoamine oxidase inactivation: From pathophysiology to therapeuticsBortolato, Marco; Chen, Kevin; Shih, Jean C.Advanced Drug Delivery Reviews (2008), 60 (13-14), 1527-1533CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)A review. Monoamine oxidases (MAOs) A and B are mitochondrial bound isoenzymes which catalyze the oxidative deamination of dietary amines and monoamine neurotransmitters, such as serotonin, norepinephrine, dopamine, β-phenylethylamine and other trace amines. The rapid degrdn. of these mols. ensures the proper functioning of synaptic neurotransmission and is critically important for the regulation of emotional behaviors and other brain functions. The byproducts of MAO-mediated reactions include several chem. species with neurotoxic potential, such as hydrogen peroxide, ammonia and aldehydes. As a consequence, it is widely speculated that prolonged excessive activity of these enzymes may be conducive to mitochondrial damages and neurodegenerative disturbances. In keeping with these premises, the development of MAO inhibitors has led to important breakthroughs in the therapy of several neuropsychiatric disorders, ranging from mood disorders to Parkinson's disease. Furthermore, the characterization of MAO knockout (KO) mice has revealed that the inactivation of this enzyme produces a no. of functional and behavioral alterations, some of which may be harnessed for therapeutic aims. In this article, we discuss the intriguing hypothesis that the attenuation of the oxidative stress induced by the inactivation of either MAO isoform may contribute to both antidepressant and antiparkinsonian actions of MAO inhibitors. This possibility further highlights MAO inactivation as a rich source of novel avenues in the treatment of mental disorders.213Laine, K.; Anttila, M.; Helminen, A.; Karnani, H.; Huupponen, R. Dose linearity study of selegiline pharmacokinetics after oral administration: evidence for strong drug interaction with female sex steroids. Br. J. Clin. Pharmacol. 1999, 47, 249– 254, DOI: 10.1046/j.1365-2125.1999.00891.x[Crossref], [PubMed], [CAS], Google Scholar213https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXisVKiu7w%253D&md5=b47fecbe9ff7e5659e5b1659370ff5f8Dose linearity study of selegiline pharmacokinetics after oral administration: evidence for strong drug interaction with female sex steroidsLaine, Kari; Anttila, Markku; Helminen, Antti; Karnani, Hari; Huupponen, RistoBritish Journal of Clinical Pharmacology (1999), 47 (3), 249-254CODEN: BCPHBM; ISSN:0306-5251. (Blackwell Science Ltd.)The purpose of this study was to characterize the dose relation of selegiline and desmethylselegiline pharmacokinetics within the selegiline dose range from 5 to 40 mg. Eight female subjects, of whom four were using oral contraceptives, ingested a single dose of 5 mg, 10 mg, 20 mg or 40 mg of selegiline HCl in an open four-period randomized study. Concns. of selegiline and desmethylselegiline in serum were measured by gas chromatog. for 5 h. As it became evident that the use of oral steroids had a drastic effect on selegiline concns., the pharmacokinetic analyses were performed sep. for oral contraceptive users and those not receiving any concomitant medication. The total AUC and Cmax of selegiline were 10-to 20-fold higher in those subjects taking oral steroids compared with subjects with no concomitant medication; this finding was consistent and statistically significant at all the four dose levels. The dose linearity of selegiline pharmacokinetics failed to be demonstrated in both groups. The AUC and Cmax of desmethylselegiline were only moderately higher (about 1.5-fold; P=NS at each dose level) in the subjects taking oral steroids than in those not receiving concomitant medication. The AUC values of desmethylselegiline increased in a dose linear manner in subjects with no concomitant medication, but not in the oral steroid group. The metabolic ratio (AUC(desmethylselegiline)/AUC(selegiline)) was several-fold lower in the group receiving oral steroids compared with the no-concomitant-medication group (at all the four dose levels). Concomitant use of oral contraceptives caused a drastic (20-fold) increase in the oral bioavailability of selegiline. The highly significant difference in the metabolic ratio between the groups provides evidence that the mechanism of the interaction between selegiline and female sex steroids involves reduced N-demethylation of selegiline. The present results suggest that concomitant use of selegiline with exogenous female sex steroids should be avoided or the dosage of selegiline should be reduced to minimize the risks of selegiline related adverse drug reactions.214Zreika, M.; Fozard, J. R.; Dudley, M. W.; Bey, P.; McDonald, I. A.; Palfreyman, M. G. MDL 72,974: a potent and selective enzyme-activated irreversible inhibitor of monoamine oxidase type B with potential for use in Parkinson’s disease. J. Neural Transm.: Parkinson's Dis. Dementia Sect. 1989, 1, 243– 254, DOI: 10.1007/BF02263478[Crossref], [PubMed], [CAS], Google Scholar214https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK3c%252FovVOnsg%253D%253D&md5=524fad71ec17bc2a9e6baadd825b07deMDL 72,974: a potent and selective enzyme-activated irreversible inhibitor of monoamine oxidase type B with potential for use in Parkinson's diseaseZreika M; Fozard J R; Dudley M W; Bey P; McDonald I A; Palfreyman M GJournal of neural transmission. Parkinson's disease and dementia section (1989), 1 (4), 243-54 ISSN:0936-3076.MDL 72,974, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine, was designed to be a selective inhibitor of monoamine oxidase type B (MAO-B). In vitro, the compound inhibits rat brain mitochondrial MAO in a concentration and time-dependent fashion and shows marked selectivity for the B form (IC50 = 680 and 3.6 nM for MAO-A and MAO-B, respectively). After oral administration to rats, the compound shows preferential inhibition of brain MAO-B with ED50 values of 8 and 0.18 mg/kg p.o. for the A and B forms, respectively. Selectivity is retained on repeat dosing. MDL 72,974 did not significantly potentiate the cardiovascular effects of intraduodenually-administered tyramine in anaesthetized rats and had only minor indirect sympathomimatic effects in the pithed rat. At MAO-B selective doses the neurotoxic effect of MPTP in mice was blocked.215Milczek, E. M.; Bonivento, D.; Binda, C.; Mattevi, A.; McDonald, I. A.; Edmondson, D. E. Structural and mechanistic studies of mofegiline inhibition of recombinant human monoamine oxidase B. J. Med. Chem. 2008, 51, 8019– 8026, DOI: 10.1021/jm8011867[ACS Full Text], [CAS], Google Scholar215https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVKlu73L&md5=a2155647066b1e971ecc2b7680ee7beeStructural and Mechanistic Studies of Mofegiline Inhibition of Recombinant Human Monoamine Oxidase BMilczek, Erika M.; Bonivento, Daniele; Binda, Claudia; Mattevi, Andrea; McDonald, Ian A.; Edmondson, Dale E.Journal of Medicinal Chemistry (2008), 51 (24), 8019-8026CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Mechanistic and structural studies have been carried out to investigate the mol. basis for the irreversible inhibition of human MAO-B by mofegiline. Competitive inhibition with substrate shows an apparent Ki of 28 nM. Irreversible inhibition of MAO-B occurs with a 1:1 M stoichiometry with no observable catalytic turnover. The absorption spectral properties of mofegiline inhibited MAO-B show features (λmax ≃ 450 nm) unlike those of traditional flavin N(5) or C(4a) adducts. Visible and near-UV CD spectra of the mofegiline-MAO-B adduct shows a neg. peak at 340 nm with an intensity similar to that of N(5) flavocyanine adducts. The x-ray crystal structure of the mofegiline-MAO-B adduct shows a covalent bond between the flavin cofactor N(5) with the distal allylamine carbon atom as well as the absence of the fluorine atom. A mechanism to explain these structural and spectral data is proposed.216Palfreyman, M. G.; McDonald, I. A.; Bey, P.; Danzin, C.; Zreika, M.; Cremer, G. Haloallylamine inhibitors of MAO and SSAO and their therapeutic potential. J. Neural. Transm. Suppl. 1994, 41, 407– 414, DOI: 10.1007/978-3-7091-9324-2_54[Crossref], [PubMed], [CAS], Google Scholar216https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXlsFalsbY%253D&md5=e3b1e61467c4cca735cb5f8d9e29e2c7Haloallylamine inhibitors of MAO and SSAO and their therapeutic potentialPalfreyman, M. G.; McDonald, I. A.; Bey, P.; Danzin, C.; Zreika, M.; Cremer, G.Journal of Neural Transmission, Supplement (1994), 41 (), 407-14CODEN: JNTSD4; ISSN:0303-6995.Based on mechanistic understandings, mol. modeling and extensive quant. structure-activity relationships, appropriately substituted haloallylamine derivs. were designed as potential mechanism-based inhibitors of MAO and/or semicarbazide-sensitive amine oxidase (SSAO). Potent inhibition of MAO-B and SSAO occurred with fluoroallylamines whereas chloroallylamines, such as MDL 72274A ((E)-2-phenyl-3-chloroallylamine hydrochloride), were selective and potent inhibitors of SSAO. MDL 72974A (E)-2-(4-fluorophenethyl)-3-fluoroallylamine hydrochloride is a potent (IC50 = 10-9M) inhibitor of both MAO-B and SSAO, with 190-fold lower affinity for MAO-A. In clin. studies, oral doses as low as 100 μg produced substantial inhibition of platelet MAO-B. Essentially complete inhibition occurred at 1 mg with the effect lasting 6-10 days. One or 4 mg MDL 72974A given daily for 28 days to 40 Parkinson's patients treated with L-dopa produced statistically significant redns. in the Unified Parkinson's Disease Rating Scale. MAO-B inhibitors, such as MDL 72974A and L-deprenyl, offer the potential of being neuroprotective in Parkinson's Disease and other neurogenerative disorders. Concomitant inhibition of SSAO may provide addnl., but as yet unproven, advantages over pure inhibitors of MAO-B.217Foot, J. S.; Deodhar, M.; Turner, C. I.; Yin, P.; van Dam, E. M.; Silva, D. G.; Olivieri, A.; Holt, A.; McDonald, I. A. The discovery and development of selective 3-fluoro-4-aryloxyallylamine inhibitors of the amine oxidase activity of semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1). Bioorg. Med. Chem. Lett. 2012, 22, 3935– 3940, DOI: 10.1016/j.bmcl.2012.04.111[Crossref], [PubMed], [CAS], Google Scholar217https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XntVyrtLg%253D&md5=cb02b80379ec2a2a6323b158f2297944The discovery and development of selective 3-fluoro-4-aryloxyallylamine inhibitors of the amine oxidase activity of semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1)Foot, Jonathan S.; Deodhar, Mandar; Turner, Craig I.; Yin, Ping; van Dam, Ellen M.; Silva, Diego G.; Olivieri, Aldo; Holt, Andrew; McDonald, Ian A.Bioorganic & Medicinal Chemistry Letters (2012), 22 (12), 3935-3940CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)A new class of 3-fluoroallyl amine-based SSAO/VAP-1 inhibitors is reported. These compds. have excellent selectivity over diamine oxidase, MAO-A and MAO-B. Synthesis and SAR studies leading to compd. 28 (PXS-4159A, I) are reported. The pharmacokinetic profile of 28 in the rat, together with activity in a murine model of lung inflammation are also disclosed.218Salmi, M.; Jalkanen, S. Cell-surface enzymes in control of leukocyte trafficking. Nat. Rev. Immunol. 2005, 5, 760– 771, DOI: 10.1038/nri1705[Crossref], [PubMed], [CAS], Google Scholar218https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtVGrurvF&md5=ca02d208fc611365ba8f7d7dcb3739c0Cell-surface enzymes in control of leukocyte traffickingSalmi, Marko; Jalkanen, SirpaNature Reviews Immunology (2005), 5 (10), 760-771CODEN: NRIABX; ISSN:1474-1733. (Nature Publishing Group)A review. Leukocyte trafficking between the blood and the tissues is pivotal for normal immune responses. Cell-adhesion mols. (such as selectins and leukocyte integrins) and chemoattractants (such as chemokines) have well-established roles in supporting leukocyte exit from the blood. Emerging data now show that, for both leukocytes and endothelial cells, enzymic reactions that are catalyzed by cell-surface-expressed enzymes with catalytic domains outside the plasma membrane (known as ectoenzymes) also make crucial contributions to this process. Ectoenzymes can function phys. as adhesion receptors and can regulate the recruitment of cells through their catalytic activities. Here, the authors provide new insights into how ectoenzymes - including nucleotidases, cyclases, ADP-ribosyltransferases, peptidases, proteases and oxidases - guide leukocyte traffic.219O’Rourke, A. M.; Wang, E. Y.; Miller, A.; Podar, E. M.; Scheyhing, K.; Huang, L.; Kessler, C.; Gao, H.; Ton-Nu, H. T.; Macdonald, M. T.; Jones, D. S.; Linnik, M. D. Anti-inflammatory effects of LJP 1586 [Z-3-fluoro-2-(4-methoxybenzyl)allylamine hydrochloride], an amine-based inhibitor of semicarbazide-sensitive amine oxidase activity. J. Pharmacol. Exp. Ther. 2008, 324, 867– 875, DOI: 10.1124/jpet.107.131672[Crossref], [PubMed], [CAS], Google Scholar219https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsFShurY%253D&md5=488fa2c75c5488de6cb038c1a253c72eAnti-inflammatory effects of LJP 1586 [Z-3-fluoro-2-(4-methoxybenzyl)allylamine hydrochloride], an amine-based inhibitor of semicarbazide-sensitive amine oxidase activityO'Rourke, Anne M.; Wang, Eric Y.; Miller, Andrew; Podar, Erika M.; Scheyhing, Kelly; Huang, Li; Kessler, Christina; Gao, Hongfeng; Ton-Nu, Huong-Thu; MacDonald, Mary T.; Jones, David S.; Linnik, Matthew D.Journal of Pharmacology and Experimental Therapeutics (2008), 324 (2), 867-875CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)Semicarbazide-sensitive amine oxidase (SSAO, amine oxidase, copper-contg. 3, and vascular adhesion protein-1) is a copper-contg. enzyme that catalyzes the oxidative deamination of primary amines to an aldehyde, ammonia, and hydrogen peroxide. SSAO is also involved in leukocyte migration to sites of inflammation, and the enzymic activity of SSAO is essential to this role. Thus, inhibition of SSAO enzyme activity represents a target for the development of small mol. anti-inflammatory compds. Here, we have characterized the novel SSAO inhibitor, Z-3-fluoro-2-(4-methoxybenzyl)allylamine hydrochloride (LJP 1586), and assessed its anti-inflammatory activity. LJP 1586 is a potent inhibitor of rodent and human SSAO activity, with IC50 values between 4 and 43 nM. The selectivity of LJP 1586 was confirmed with a broad panel of receptors and enzymes that included the monoamine oxidases A and B. Oral administration of LJP 1586 resulted in complete inhibition of rat lung SSAO, with an ED50 between 0.1 and 1 mg/kg, and a pharmacodynamic half-life of greater than 24 h. In a mouse model of inflammatory leukocyte trafficking oral dosing with LJP 1586 resulted in significant dose-dependent inhibition of neutrophil accumulation, with an effect comparable to that of anti-leukocyte function-assocd. antigen-1 antibody. In a rat model of LPS-induced lung inflammation, administration of 10 mg/kg LJP 1586 resulted in a 55% significant redn. in transmigrated cells recovered by bronchoalveolar lavage. The results demonstrate that a selective, orally active small mol. inhibitor of SSAO is an effective anti-inflammatory compd. in vivo and provide further support for SSAO as a therapeutic anti-inflammatory target.220Dow, J.; Piriou, F.; Wolf, E.; Dulery, B. D.; Haegele, K. D. Novel carbamate metabolites of mofegiline, a primary amine monoamine oxidase B inhibitor, in dogs and humans. Drug Metab. Dispos. 1994, 22, 738– 749[PubMed], [CAS], Google Scholar220https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXmslWhs78%253D&md5=b437862f1906cba7f30bbc0a633c3ff6Novel carbamate metabolites of mofegiline, a primary amine monoamine oxidase B inhibitor, in dogs and humansDow, James; Piriou, Francois; Wolf, Evelyne; Dulery, Bertrand D.; Haegele, Klaus D.Drug Metabolism and Disposition (1994), 22 (5), 738-49CODEN: DMDSAI; ISSN:0090-9556.Mofegiline or MDL 72,974A ((E)-4-fluoro-β-fluoromethylene benzene butanamine hydrochloride) is a selective enzyme-activated irreversible inhibitor of monoamine oxidase B, which is under development for use in the treatment of Parkinson's disease. Male beagle dogs were given single oral (20 mg/kg) and i.v. (5 mg/kg) doses of [14C]-Mofegiline. Total radioactivity excreted in urine and feces over 96 h was, resp., 75.5 and 6.3% of the dose after oral and 67.9 and 3.9% after i.v. administration. Unchanged drug in urine represented 3% of the dose after oral and less than 1% after i.v. administration. Mofegiline was thus extensively metabolized in dogs, and urinary excretion was the major route of elimination of metabolites. HPLC, with online radioactivity detection, showed the presence of four major peaks (M1, M2, M3, and M4), representing resp. 50, 9, 5, and 0.5% of the administered dose excreted in 0.24 h urine. TSP-LC-MS, Fab-MS, and NMR spectra of the purified metabolites were obtained. M1, the major metabolite in dogs, was shown to have undergone defluorination of the β-fluoromethylene moiety, and one carbon addn. Its structure was confirmed to be a cyclic carbamate. M2 was a N-carbamoyl-O-β-D-glucuronide conjugate of parent drug. The formation of M1 and M2 is likely to involve initial reversible addn. of CO2 to the primary amine function. M3 was a N-succinyl conjugate of the parent drug. M4 had also undergone defluorination to yield a urea adduct of an unsatd. α,β aldehyde. Structures of M1 and M3 were further confirmed by comparing their MS and NMR spectra with those of authentic ref. compds. TSP-LC-MS ion chromatographs of human urine, obtained from two male volunteers after oral administration of 24 mg of drug, showed selected mol. ion peaks with the same retention time as the metabolites identified in dogs. In humans, these common metabolites represented a similar percentage of the administered dose to that in dogs. The present study demonstrates that NMR, TSP-LC-MS, and FAB-MS are complementary anal. techniques, which allow structural identification of unhydrolyzed drug conjugates. The formation of carbamates of amine-contg. drugs may be more common than previously reported.221Pan, Y.; Gerasimov, M. R.; Kvist, T.; Wellendorph, P.; Madsen, K. K.; Pera, E.; Lee, H.; Schousboe, A.; Chebib, M.; Brauner-Osborne, H.; Craft, C. M.; Brodie, J. D.; Schiffer, W. K.; Dewey, S. L.; Miller, S. R.; Silverman, R. B. (1S, 3S)-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (CPP-115), a potent γ-aminobutyric acid aminotransferase inactivator for the treatment of cocaine addiction. J. Med. Chem. 2012, 55, 357– 366, DOI: 10.1021/jm201231w[ACS Full Text], [CAS], Google Scholar221https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFGmt7jN&md5=70944b375478c340c8d0c999317b4293(1S, 3S)-3-Amino-4-difluoromethylenyl-1-cyclopentanoic Acid (CPP-115), a Potent γ-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Cocaine AddictionPan, Yue; Gerasimov, Madina R.; Kvist, Trine; Wellendorph, Petrine; Madsen, Karsten K.; Pera, Elena; Lee, Hyunbeom; Schousboe, Arne; Chebib, Mary; Brauner-Osborne, Hans; Craft, Cheryl M.; Brodie, Jonathan D.; Schiffer, Wynne K.; Dewey, Stephen L.; Miller, Steven R.; Silverman, Richard B.Journal of Medicinal Chemistry (2012), 55 (1), 357-366CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Vigabatrin, a GABA aminotransferase (GABA-AT) inactivator, is used to treat infantile spasms and refractory complex partial seizures and is in clin. trials to treat addiction. We evaluated a novel GABA-AT inactivator (1S, 3S)-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (CPP-115, compd. 1) and obsd. that it does not exhibit other GABAergic or off-target activities and is rapidly and completely orally absorbed and eliminated. By use of in vivo microdialysis techniques in freely moving rats and microPET imaging techniques, 1 produced similar inhibition of cocaine-induced increases in extracellular dopamine and in synaptic dopamine in the nucleus accumbens at 1/300 to 1/600 the dose of vigabatrin. It also blocks expression of cocaine-induced conditioned place preference at a dose 1/300 that of vigabatrin. Electroretinog. (ERG) responses in rats treated with 1, at doses 20-40 times higher than those needed to treat addiction in rats, exhibited redns. in ERG responses, which were less than the redns. obsd. in rats treated with vigabatrin at the same dose needed to treat addiction in rats. In conclusion, 1 can be administered at significantly lower doses than vigabatrin, which suggests a potential new treatment for addiction with a significantly reduced risk of visual field defects.222Silverman, R. B. Design and mechanism of GABA aminotransferase inactivators. Treatments for epilepsies and addictions. Chem. Rev. 2018, 118, 4037– 4070, DOI: 10.1021/acs.chemrev.8b00009[ACS Full Text], [CAS], Google Scholar222https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXlt1yhu7k%253D&md5=0071d0e7668f05d37db995bb10430c16Design and Mechanism of GABA Aminotransferase Inactivators. Treatments for Epilepsies and AddictionsSilverman, Richard B.Chemical Reviews (Washington, DC, United States) (2018), 118 (7), 4037-4070CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. When the brain concn. of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) diminishes below a threshold level, the excess neuronal excitation can lead to convulsions. This imbalance in neurotransmission can be cor. by inhibition of the enzyme γ-aminobutyric acid aminotransferase (GABA-AT), which catalyzes the conversion of GABA to the excitatory neurotransmitter L-glutamic acid. It also has been found that raising GABA levels can antagonize the rapid elevation and release of dopamine in the nucleus accumbens, which is responsible for the reward response in addiction. Therefore, the design of new inhibitors of GABA-AT, which increases brain GABA levels, is an important approach to new treatments for epilepsy and addiction. This review summarizes findings over the last 40 or so years of mechanism-based inactivators (unreactive compds. that require the target enzyme to catalyze their conversion to the inactivating species, which inactivate the enzyme prior to their release) of GABA-AT with emphasis on their catalytic mechanisms of inactivation, presented according to org. chem. mechanism, with minimal pharmacol., except where important for activity in epilepsy and addiction. Patents, abstrs., and conference proceedings are not covered in this review. The inactivation mechanisms described here can be applied to the inactivations of a wide variety of unrelated enzymes.223Lippert, B.; Metcalf, B. W.; Jung, M. J.; Casara, P. 4-Amino-hex-5-enoic acid, a selective catalytic inhibitor of 4-aminobutyric-acid aminotransferase in mammalian brain. Eur. J. Biochem. 1977, 74, 441– 445, DOI: 10.1111/j.1432-1033.1977.tb11410.x[Crossref], [PubMed], [CAS], Google Scholar223https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2sXktVWlu7o%253D&md5=849abfb7d249e7e40457c38e3ddfb0ff4-Amino-hex-5-enoic acid, a selective catalytic inhibitor of 4-aminobutyric-acid aminotransferase in mammalian brainLippert, Bruce; Metcalf, Brian W.; Jung, Michel J.; Casara, PatrickEuropean Journal of Biochemistry (1977), 74 (3), 441-5CODEN: EJBCAI; ISSN:0014-2956.Incubation of rat brain 4-aminobutyrate (I) aminotransferase (II) with 4-amino-hex-5-enoic acid (III), an analog of I, results in a time-dependent irreversible loss of activity. In the presence of 0.1 mM III, the half-life of the inactivation process is approx. 6 min. Low concns. of L-glutamic acid or I protect against this inactivation, while 2-oxoglutarate prevents this protection, suggesting that only the pyridoxal form of II is susceptible to inhibition by III. The irreversible inhibition of mammalian II by III is selective. There is no inhibition of II from Pseudomonas fluorescens with III at mM concns. Even at 10 mM, there is no irreversible inhibition of mammalian glutamate decarboxylase or of aspartate aminotransferase, while alanine aminotransferase is inhibited >500-fold more slowly than II.224Storici, P.; De Biase, D.; Bossa, F.; Bruno, S.; Mozzarelli, A.; Peneff, C.; Silverman, R. B.; Schirmer, T. Structures of γ-aminobutyric acid (GABA) aminotransferase, a pyridoxal 5′-phosphate, and [2Fe-2S] cluster-containing enzyme, complexed with γ-ethynyl-GABA and with the antiepilepsy drug vigabatrin. J. Biol. Chem. 2004, 279, 363– 373, DOI: 10.1074/jbc.M305884200[Crossref], [PubMed], [CAS], Google Scholar224https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhtVSqtrfK&md5=5c0b22682c6419f86551270df45cb6d7Structures of γ-aminobutyric acid (GABA) aminotransferase, a pyridoxal 5'-phosphate, and [2Fe-2S] cluster-containing enzyme, complexed with γ-ethynyl-GABA and with the antiepilepsy drug vigabatrinStorici, Paola; De Biase, Daniela; Bossa, Francesco; Bruno, Stefano; Mozzarelli, Andrea; Peneff, Caroline; Silverman, Richard B.; Schirmer, TilmanJournal of Biological Chemistry (2004), 279 (1), 363-373CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)γ-Aminobutyrate (GABA) aminotransferase (I) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme responsible for the degrdn. of the inhibitory neurotransmitter, GABA. I is a validated target for antiepilepsy drugs because its selective inhibition raises GABA concns. in brain. The antiepilepsy drug, vigabatrin (γ-vinyl-GABA), was investigated in the past by various biochem. methods and these resulted in several proposals for its mechanisms of inactivation. Here, the authors solved and compared the crystal structures of pig liver I in its native form (to 2.3-Å resoln.) and its complex with vigabatrin as well as with its close analog, γ-ethynyl-GABA (to 2.3 and 2.8 Å, resp.). Both inactivators formed a covalent ternary adduct with the active site Lys-329 residue and the PLP cofactor. The crystal structures provided direct support for specific inactivation mechanisms proposed earlier on the basis of radiolabeling expts. The reactivity of I crystals with the 2 GABA analogs was also investigated by polarized absorption microspectrophotometry. The spectral data were discussed in relation to the proposed mechanism. Intriguingly, all 3 structures revealed a [2Fe-2S] cluster of yet unknown function at the center of the dimeric mol. in the vicinity of the PLP cofactor.225Pan, Y.; Qiu, J.; Silverman, R. B. Design, synthesis, and biological activity of a difluoro-substituted, conformationally rigid vigabatrin analogue as a potent γ-aminobutyric acid aminotransferase inhibitor. J. Med. Chem. 2003, 46, 5292– 5293, DOI: 10.1021/jm034162s[ACS Full Text], [CAS], Google Scholar225https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXovVClt7k%253D&md5=30a2fb61e2beb595154f7a9519188d63Design, Synthesis, and Biological Activity of a Difluoro-Substituted, Conformationally Rigid Vigabatrin Analogue as a Potent γ-Aminobutyric Acid Aminotransferase InhibitorPan, Yue; Qiu, Jian; Silverman, Richard B.Journal of Medicinal Chemistry (2003), 46 (25), 5292-5293CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Previously it was found that a conformationally rigid analog [(1R,4S)-4-amino-2-cyclopentene-1-carboxylic acid] of the epilepsy drug (S)-vigabatrin did not inactivate γ-aminobutyric acid aminotransferase (GABA-AT). Ring opening of (1S,4S)-6-methylene-2-(phenylmethyl)-2-azabicyclo[2.2.1]heptan-3-one gave (1S,4S)-4-amino-3-methylene-1-cyclopentanecarboxylic acid (I). I, having an exocyclic double bond, was found to inactivate GABA-AT, but only in the absence of 2-mercaptoethanol. The corresponding difluoro-substituted analog [(+)-(1S,4S)-4-amino-3-(difluoromethylene)-1-cyclopentanecarboxylic acid] was synthesized and was shown to be a very potent time-dependent inhibitor, even in the presence of 2-mercaptoethanol.226Lee, H.; Doud, E. H.; Wu, R.; Sanishvili, R.; Juncosa, J. I.; Liu, D.; Kelleher, N. L.; Silverman, R. B. Mechanism of inactivation of γ-aminobutyric acid aminotransferase by (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115). J. Am. Chem. Soc. 2015, 137, 2628– 2640, DOI: 10.1021/ja512299n[ACS Full Text], [CAS], Google Scholar226https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVWgt7k%253D&md5=518c957dfdcd01be2a8f3b2e3ab2f14aMechanism of inactivation of γ-aminobutyric acid aminotransferase by (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115)Lee, Hyunbeom; Doud, Emma H.; Wu, Rui; Sanishvili, Ruslan; Juncosa, Jose I.; Liu, Dali; Kelleher, Neil L.; Silverman, Richard B.Journal of the American Chemical Society (2015), 137 (7), 2628-2640CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)γ-Aminobutyrate (GABA) aminotransferase (I) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concn. of GABA falls below a threshold level, convulsions can occur. Inhibition of I raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurol. disorders, including drug addiction. Among the analogs that the authors previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115), showed 187-fold greater potency than that of vigabatrin, a known inactivator of I and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently, CPP-115 was shown to have no adverse effects in a phase I clin. trial. Here, the authors report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes I-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of 2 F- ions and coenzyme conversion to pyridoxamine 5'-phosphate (PMP). The partition ratio for CPP-115 with pig brain I was ∼2000, releasing cyclopentanone-2,4-dicarboxylateand 2 other precursors of this compd. Time-dependent inactivation occurred by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP, which disrupted an electrostatic interaction between Glu-270 and Arg-445 to form an electrostatic interaction between Arg-445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. This represents a novel mechanism for inactivation of I and a new approach for the design of mechanism-based inactivators in general.227Jeschke, P.; Baston, E.; Leroux, F. R. α-Fluorinated ethers as “exotic” entity in medicinal chemistry. Mini-Rev. Med. Chem. 2007, 7, 1027– 1034, DOI: 10.2174/138955707782110150[Crossref], [PubMed], [CAS], Google Scholar227https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1Sjs7fN&md5=ad28d99858f35257c9bec151e6a4db9aα-Fluorinated ethers as "exotic" entity in medicinal chemistryJeschke, Peter; Baston, Eckhard; Leroux, Frederic R.Mini-Reviews in Medicinal Chemistry (2007), 7 (10), 1027-1034CODEN: MMCIAE; ISSN:1389-5575. (Bentham Science Publishers Ltd.)A review. After nitrogen, fluorine occupies the position of second favorite heteroelement in life science-oriented research. In contrast, the trifluoromethoxy group is still perhaps the least well understood fluorine substituent, although its occurrence has significantly increased in the recent years. Today, significant application areas for trifluoromethoxy substituted pharmaceuticals are in the field of analgesics, anesthetics, cardiovascular drugs, respiratory drugs, psychopharmacol. drugs, neurol. drugs, gastrointestinal drugs and anti-infective therapeutics. The present review will give an overlook of its use in medicinal chem.228Logvinenko, I. G.; Markushyna, Y.; Kondratov, I. S.; Vashchenko, B. V.; Kliachyna, M.; Tokaryeva, Y.; Pivnytska, V.; Grygorenko, O. O.; Haufe, G. Synthesis, physico-chemical properties and microsomal stability of compounds bearing aliphatic trifluoromethoxy group. J. Fluorine Chem. 2020, 231, 109461, DOI: 10.1016/j.jfluchem.2020.109461[Crossref], [CAS], Google Scholar228https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXivVens7c%253D&md5=283bf1da7c5138c4a6d4b7df6eb8bdb6Synthesis, physico-chemical properties and microsomal stability of compounds bearing aliphatic trifluoromethoxy groupLogvinenko, Ivan G.; Markushyna, Yevheniia; Kondratov, Ivan S.; Vashchenko, Bohdan V.; Kliachyna, Maria; Tokaryeva, Yuliya; Pivnytska, Valentyna; Grygorenko, Oleksandr O.; Haufe, GunterJournal of Fluorine Chemistry (2020), 231 (), 109461CODEN: JFLCAR; ISSN:0022-1139. (Elsevier B.V.)Effects of the trifluoromethoxy substituent on physico-chem. properties of compds., such as kinetic soly., lipophilicity and microsomal clearance, was studied in a series of aliph. derivs. It was found that kinetic soly. of the CF3O-contg. compds. was comparable to that of analogs, i.e. compds. bearing CH3O and CF3 moieties. The CF3O-substituted compds. had higher lipophilicity as compared to methoxy analogs, and nearly the same like CF3-bearing compds. Microsomal stability studies indicated that the trifluoromethoxy group typically decreased metabolic stability of the corresponding derivs. as compared to either CH3O- or CF3-substituted counterparts, except for N-alkoxy(sulfon)amide series.229Trachsel, D. Fluorine in psychedelic phenethylamines. Drug Test. Anal. 2012, 4, 577– 590, DOI: 10.1002/dta.413[Crossref], [PubMed], [CAS], Google Scholar229https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XivVGhu7s%253D&md5=a9609e3ef07f65c2bb5778d04352bf6cFluorine in psychedelic phenethylaminesTrachsel, DanielDrug Testing and Analysis (2012), 4 (7-8), 577-590CODEN: DTARBG; ISSN:1942-7603. (John Wiley & Sons Ltd.)A review. The so-called psychedelic phenethylamines represent a class of drugs with a large range of psychoactive properties in humans, ranging from naturally occurring mescaline to amphetamine analogs and homologues. The interest in many of these compds., occasionally referred to as designer-drugs, is widely dispersed across popular culture and political and scientific communities. In recent decades, fluorine has become a powerful and important tool in medicinal chem. In addn., fluorine-contg. compds. and medicines can be found in numerous com. successful pharmaceuticals that have gained a market share of some 5-15%. One might anticipate this trend to increase in the future. As far as fluorinated phenethylamines are concerned, much less is known about their chem. and pharmacol. This paper provides an overview regarding the biol. properties of over 60 fluorinated phenethylamines and discusses both historical and recent chem.-related developments. It was shown that the introduction of fluorine into the phenethylamine nucleus can impact greatly on psychoactivity of these compds., ranging from marked loss to enhancement and prolongation of effects. For example, in contrast to the psychoactive escaline (70), it was obsd. that its fluoroescaline (76) counterpart was almost devoid of psychoactive effects. Difluoroescaline (77), on the other hand, retained, and trifluoroescaline (78) showed increased human potency of escaline (70). Difluoromescaline (72) and trifluoromescaline (73) increasingly surpassed human potency and duration of mescaline (22) effects. Copyright © 2012 John Wiley & Sons, Ltd.230Laurence, C.; Brameld, K. A.; Graton, J.; Le Questel, J. Y.; Renault, E. The pK(BHX) database: toward a better understanding of hydrogen-bond basicity for medicinal chemists. J. Med. Chem. 2009, 52, 4073– 4086, DOI: 10.1021/jm801331y[ACS Full Text], [CAS], Google Scholar230https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXnsFKns7w%253D&md5=9ce4bea3e810752baba6ea5784b55ea0The pKBHX Database: Toward a Better Understanding of Hydrogen-Bond Basicity for Medicinal ChemistsLaurence, Christian; Brameld, Ken A.; Graton, Jerome; Le Questel, Jean-Yves; Renault, EricJournal of Medicinal Chemistry (2009), 52 (14), 4073-4086CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. The pKBHX database is presented, which corresponds to three main fields of data: hydrogen bond donor identification, thermodn. data and spectroscopic data. The pKBHX scale of hydrogen donor basicity differs considerable from the pKBH+ scale of proton transfer basicity. The hydrogen bond basicities of functional groups relevant to medicinal chem. and drug design is reviewed.231Xing, L.; Blakemore, D. C.; Narayanan, A.; Unwalla, R.; Lovering, F.; Denny, R. A.; Zhou, H.; Bunnage, M. E. Fluorine in drug design: a case study with fluoroanisoles. ChemMedChem 2015, 10, 715– 726, DOI: 10.1002/cmdc.201402555[Crossref], [PubMed], [CAS], Google Scholar231https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkvVWku7Y%253D&md5=45dbead703594c31581cc6c7e62d1b81Fluorine in Drug Design: A Case Study with FluoroanisolesXing, Li; Blakemore, David C.; Narayanan, Arjun; Unwalla, Ray; Lovering, Frank; Denny, R. Aldrin; Zhou, Huanyu; Bunnage, Mark E.ChemMedChem (2015), 10 (4), 715-726CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)Anisole and fluoroanisoles display distinct conformational preferences, as evident from a survey of their crystal structures. In addn. to altering the free ligand conformation, various degrees of fluorination have a strong impact on physicochem. and pharmacokinetic properties. Anal. of anisole and fluoroanisole matched mol. pairs in the Pfizer corporate database reveals interesting trends: (1) PhOCF3 increases log D by ∼1 log unit over PhOCH3 compds.; (2) PhOCF3 shows lower passive permeability despite its higher lipophilicity; and (3) PhOCF3 does not appreciably improve metabolic stability over PhOCH3. Emerging from the investigation, difluoroanisole (PhOCF2H) strikes a better balance of properties with noticeable advantages of log D and transcellular permeability over PhOCF3. Synthetic assessment illustrates that the routes to access difluoroanisoles are often more straightforward than those for trifluoroanisoles. Whereas replacing PhOCH3 with PhOCF3 is a common tactic to optimize ADME properties, the anal. suggests PhOCF2H may be a more attractive alternative, and greater exploitation of this motif is recommended.232Federsel, D.; Herrmann, A.; Christen, D.; Sander, S.; Willner, H.; Oberhammer, H. Structure and conformation of α,α,α-trifluoroanisol, C6H5OCF3. J. Mol. Struct. 2001, 567–568, 127– 136, DOI: 10.1016/S0022-2860(01)00541-5[Crossref], [CAS], Google Scholar232https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXlvF2isb0%253D&md5=4b1903e5580b8440c797d4e9031d5da4Structure and conformation of α,α,α-trifluoroanisole, C6H5OCF3Federsel, Daniela; Herrmann, Angelika; Christen, Dines; Sander, Stefan; Willner, Helge; Oberhammer, HeinzJournal of Molecular Structure (2001), 567-568 (), 127-136CODEN: JMOSB4; ISSN:0022-2860. (Elsevier Science B.V.)The geometric structure of α,α,α-trifluoroanisole, C6H5OCF3, was studied by gas electron diffraction (GED), microwave spectroscopy (MW), matrix IR spectroscopy and quantum chem. methods. From the three exptl. techniques, we conclude that only the perpendicular conformer (O-CF3 bond perpendicular to the benzene plane) is present in the gas-phase, although a slightly better fit of the GED intensities is obtained for a small contribution (12(8)%) of the planar form. Thus, the orientation of the O-CH3 bond in anisole changes from planar to perpendicular upon fluorination of the Me group. The predictions of quantum chem. calcns. for the conformational properties depend on the computational method and on the size of basis sets. HF/6-31G* and MP2 (6-31G* or 6-311(2d) basis sets) calcns. predict the existence of a single conformer with perpendicular orientation, in agreement with our exptl. data. The B3LYP method, however, results in a mixt. of both conformers and HF/3-21G* calcns. predict a single conformer with intermediate orientation.233Klocker, J.; Karpfen, A.; Wolschann, P. On the structure and torsional potential of trifluoromethoxybenzene: an ab initio and density functional study. Chem. Phys. Lett. 2003, 367, 566– 575, DOI: 10.1016/S0009-2614(02)01786-4[Crossref], [CAS], Google Scholar233https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XptlWgt70%253D&md5=bb476890fdb2a1e48ffdbe9966d8515aOn the structure and torsional potential of trifluoromethoxybenzene: an ab initio and density functional studyKlocker, Johanna; Karpfen, Alfred; Wolschann, PeterChemical Physics Letters (2002), 367 (5,6), 566-575CODEN: CHPLBC; ISSN:0009-2614. (Elsevier Science B.V.)The torsional potential of trifluoromethoxybenzene around the aryl-O bond was investigated with the aid of large-scale ab initio calcns. performed at the Moller-Plesset second order (MP2) level, with several post-MP2 methods, and with a hybrid d. functional method (B3LYP). Contrary to several recent reports, we do not find substantial qual. differences between MP2 and B3LYP results, provided sufficiently large basis sets are used. The results are confronted with analogous MP2 and B3LYP data for methoxybenzene, for hypothetical anions as obtained by deprotonation at the para-position, and for ethylbenzene. The trends in the calcd. torsional potentials, barrier heights and energy differences between conformers are discussed and correlated with selected structural parameters.234Kuo, E. A.; Hambleton, P. T.; Kay, D. P.; Evans, P. L.; Matharu, S. S.; Little, E.; McDowall, N.; Jones, C. B.; Hedgecock, C. J.; Yea, C. M.; Chan, A. W.; Hairsine, P. W.; Ager, I. R.; Tully, W. R.; Williamson, R. A.; Westwood, R. Synthesis, structure-activity relationships, and pharmacokinetic properties of dihydroorotate dehydrogenase inhibitors: 2-cyano-3-cyclopropyl-3-hydroxy-N-[3′-methyl-4’-(trifluoromethyl)phenyl ] propenamide and related compounds. J. Med. Chem. 1996, 39, 4608– 4621, DOI: 10.1021/jm9604437[ACS Full Text], [CAS], Google Scholar234https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xmt1ylsLo%253D&md5=1bffdde8bb8429fd7d309f7bb26660bcSynthesis, Structure-Activity Relationships, and Pharmacokinetic Properties of Dihydroorotate Dehydrogenase Inhibitors: 2-Cyano-3-cyclopropyl-3-hydroxy- N-[3'-methyl-4'-(trifluoromethyl)phenyl]propenamide and Related CompoundsKuo, Elizabeth A.; Hambleton, Philip T.; Kay, David P.; Evans, Phillip L.; Matharu, Saroop S.; Little, Edward; McDowall, Neil; Jones, C. Beth; Hedgecock, Charles J. R.; et al.Journal of Medicinal Chemistry (1996), 39 (23), 4608-4621CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The active, ring-opened metabolite of the novel immunosuppressive agent leflunomide has been shown to inhibit the enzyme dihydroorotate dehydrogenase (DHODH). This enzyme catalyzes the fourth step in de novo pyrimidine biosynthesis. A series of analogs of the leflunomide metabolite have been synthesized. Their in vivo biol. activity detd. in rat and mouse delayed type hypersensitivity has been found to correlate well with their in vitro DHODH potency. The most promising compd. has shown activity in rat and mouse collagen (II)-induced arthritis models (ED50 = 2 and 31 mg/kg, resp.) and has shown a shorter half-life in man when compared with leflunomide. Clin. studies in rheumatoid arthritis are in progress.235Dihel, L.; Kittleson, C.; Mulvihill, K.; Johnson, W. W. Oxidative metabolism of the trifluoromethoxy moiety of OSI-930. Drug Metab. Drug Interact. 2009, 24, 95– 121, DOI: 10.1515/DMDI.2009.24.2-4.95[Crossref], [PubMed], [CAS], Google Scholar235https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXktlOqu7o%253D&md5=8896ae50e1ffda023f083637e4bf859dOxidative metabolism of the trifluoromethoxy moiety of OSI-930Dihel, Larry; Kittleson, Christine; Mulvihill, Kristen; Johnson, William W.Drug Metabolism and Drug Interactions (2009), 24 (2-4), 95-121CODEN: DMDIEQ; ISSN:0792-5077. (Freund Publishing House Ltd.)Cytochrome P 450 can catalyze a wide array of remarkable oxidns., including O-dealkylations, which are performed via oxidn. of the α-carbon of the ether. When C-H bonds are replaced with C-F bonds, however, the bond strength is much greater, and it significantly deters oxidn. at the carbon. Another recently elucidated reaction catalyzed by P 450, ipso substitution, results in displacement of arom. ring substituents such as an alkoxy group via hydroxyl substitution. Through LC/MS/MS, we show the CYP-mediated oxidative displacement of the trifluoromethoxy group from the Ph constituent in OSI-930, a novel small mol. c-Kit/VEGF-r inhibitor in clin. studies to treat cancer. Based on C-F bond strength, reported phenacetin studies, and α-quaternary alkylphenol studies, we propose an ipso-substitution mechanism for this oxidative biotransformation. In vivo, this hydroxylated metabolite goes on to form the ether conjugate with glucuronide.236Hinson, J. A.; Nelson, S. D.; Gillette, J. R. Metabolism of [p-18O]-phenacetin: the mechanism of activation of phenacetin to reactive metabolites in hamsters. Mol. Pharmacol. 1979, 15, 419– 427[PubMed], [CAS], Google Scholar236https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1MXktlamur0%253D&md5=282d8fcd9d48a3d65597a407f3baafb7Metabolism of [p-18O]-phenacetin: the mechanism of activation of phenacetin to reactive metabolites in hamstersHinson, Jack A.; Nelson, Sidney D.; Gillette, James R.Molecular Pharmacology (1979), 15 (2), 419-27CODEN: MOPMA3; ISSN:0026-895X.There are 3 different pathways by which phenacetin (I) [62-44-2] can be converted by liver enzymes to electrophilic reactive metabolites: (1) I is first deethylated to acetaminophen [103-90-2] which is subsequently activated by cytochrome P-450 [9035-51-2], (2) I is activated via an intermediate, (3) I is first converted to N-hydroxyphenacetin [19315-64-1], then activated by sulfation or glucuronidation. These 3 pathways were distinguished by the disposition of 18O in the para position of the ring; (1) when acetaminophen-4-18O was activated in vitro and in vivo there was a negligible loss of 18O in the resp. acetaminophen-glutathione conjugate and the urinary mercapturic acid [616-91-1]; (2) when I-4-18O wa activated in vitro there was a 50% loss of 18O in the acetaminophen-glutathione conjugate; (3) when N-hydroxyphenacetin glucuronide [69783-19-3] was incubated with H218O there was a quant. incorporation of 18O into the acetaminophen-glutathione conjugate. When N-hydroxyacetaminophen [70110-93-9], the proposed intermediate in pathway 1, was incubated with glutathione and H218O there was no incorporation of 18O into the acetaminophen-glutathione conjugate. The relative in vivo importance of the 3 pathways was investigated by i.p. injection of 50 mg/kg of I-4-18O into hamsters. The urinary mercapturic acid showed ∼10% loss of 18O label, indicating that deethylation of I to acetaminophen followed by activation of acetaminophen. Thus pathway 1 appears to be the predominant pathway in vivo.237Rietjens, I. M.; den Besten, C.; Hanzlik, R. P.; van Bladeren, P. J. Cytochrome P450-catalyzed oxidation of halobenzene derivatives. Chem. Res. Toxicol. 1997, 10, 629– 635, DOI: 10.1021/tx9601061[ACS Full Text], [CAS], Google Scholar237https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXjs1Wgt74%253D&md5=54b2699f7df748a5a507007040bf94adCytochrome P450-Catalyzed Oxidation of Halobenzene DerivativesRietjens, Ivonne M. C. M.; den Besten, Cathaline; Hanzlik, Robert P.; van Bladeren, Peter J.Chemical Research in Toxicology (1997), 10 (6), 629-635CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)A review and discussion with refs. Multiple pathways in the generation of (proximate) toxic metabolites of a halobenzene, alkylating properties of epoxides and quinones, mechanism of benzoquinone formation, role of epoxides in the pathway to phenolic metabolites are discussed.238National Library of Medicine. https://pubchem.ncbi.nlm.nih.gov/compound/Carbonyl-fluoride (accessed 2020-03-14).239Deinum, J.; Mattsson, C.; Inghardt, T.; Elg, M. Biochemical and pharmacological effects of the direct thrombin inhibitor AR-H067637. Thromb. Haemostasis 2009, 101, 1051– 1059, DOI: 10.1160/TH08-09-0586[Crossref], [PubMed], [CAS], Google Scholar239https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXos1aktbo%253D&md5=01ec6f363cff05b7389618e4e406a8eaBiochemical and pharmacological effects of the direct thrombin inhibitor AR-H067637Deinum, Johanna; Mattsson, Christer; Inghardt, Tord; Elg, MargaretaThrombosis and Haemostasis (2009), 101 (6), 1051-1059CODEN: THHADQ; ISSN:0340-6245. (Schattauer GmbH)AZD0837 is in development as a new oral anticoagulant for use in thromboembolic disorders. In vivo, AZD0837 is converted to AR-H067637, a selective and reversible direct thrombin inhibitor. Established biochem. methods were used to assess and measure the biochem. and pharmacol. properties of AR-H067637. Both direct Biacore binding studies of AR-H067637 with immobilized α-thrombin and inhibition studies using pre-steady state kinetics with thrombin in the fluid phase confirmed that AR-H067637 is a rapid-binding, reversible and potent (inhibition const. Ki = 2-4 nM), competitive inhibitor of thrombin, as well as of thrombin bound to fibrin (clotbound thrombin) or to thrombomodulin. The total amt. of free thrombin generated in platelet-poor clotting plasma was inhibited concn.-dependently by AR-H067637, with a concn. giving half maximal inhibition (IC50) of 0.6 μM. Moreover, AR-H067637 is, with the exception of trypsin, a selective inhibitor for thrombin without inhibiting other serine proteases involved in haemostasis. Furthermore, no anticoagulant effect of the prodrug was found AR-H067637 prolonged the clotting time concn.-dependently in a range of plasma coagulation assays including activated partial thromboplastin time, prothrombin time, prothrombinase-induced clotting time, thrombin time and ecarin clotting time. The two latter assays were found to be most sensitive for assessing the anticoagulant effect of AR-H067637 (plasma IC50 93 and 220 nM, resp.). AR-H067637 also inhibited thrombin-induced platelet activation (by glycoprotein Ilb/Illa exposure, IC50 8.4 nM) and aggregation (IC50 0.9 nM). In conclusion, AR-H067637 is a selective, reversible, competitive inhibitor of α-thrombin, with a predictable anticoagulant effect demonstrated in plasma coagulation assays.240Malamas, M. S.; Robichaud, A.; Erdei, J.; Quagliato, D.; Solvibile, W.; Zhou, P.; Morris, K.; Turner, J.; Wagner, E.; Fan, K.; Olland, A.; Jacobsen, S.; Reinhart, P.; Riddell, D.; Pangalos, M. Design and synthesis of aminohydantoins as potent and selective human β-secretase (BACE1) inhibitors with enhanced brain permeability. Bioorg. Med. Chem. Lett. 2010, 20, 6597– 6605, DOI: 10.1016/j.bmcl.2010.09.029[Crossref], [PubMed], [CAS], Google Scholar240https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlSmsL7I&md5=848a1bfa79278b0c7a72749f9be45ad8Design and synthesis of aminohydantoins as potent and selective human β-secretase (BACE1) inhibitors with enhanced brain permeabilityMalamas, Michael S.; Robichaud, Albert; Erdei, Jim; Quagliato, Dominick; Solvibile, William; Zhou, Ping; Morris, Koi; Turner, Jim; Wagner, Erik; Fan, Kristi; Olland, Andrea; Jacobsen, Steve; Reinhart, Peter; Riddell, David; Pangalos, MenelasBioorganic & Medicinal Chemistry Letters (2010), 20 (22), 6597-6605CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)The identification of small mol. aminohydantoins as potent and selective human β-secretase inhibitors is reported. These analogs exhibit good brain permeability (40-70%), low nanomolar potency for BACE1, and demonstrate >100-fold selectivity for the structurally related aspartyl proteases cathepsin D, renin and pepsin. Alkyl and alkoxy groups at the meta-position of the P1 Ph, which extend toward the S3 region of the enzyme, have contributed to the ligand's reduced affinity for the efflux transporter protein P-gp, and decreased topol. polar surface area, thus resulting in enhanced brain permeability. A fluorine substitution at the para-position of the P1 Ph has contributed to 100-fold decrease of CYP3A4 inhibition and enhancement of compd. metabolic stability. The plasma and brain protein binding properties of these new analogs are affected by substitutions at the P1 Ph moiety. Higher compd. protein binding was obsd. in the brain than in the plasma. Two structurally diverse potent BACE1 inhibitors (84 and 89) reduced 30% plasma Aβ40 in the Tg2576 mice in vivo model at 30 mg/kg po.241Williams, S. J.; Zammit, S. C.; Cox, A. J.; Shackleford, D. M.; Morizzi, J.; Zhang, Y.; Powell, A. K.; Gilbert, R. E.; Krum, H.; Kelly, D. J. 3′,4’-Bis-difluoromethoxycinnamoylanthranilate (FT061): an orally-active antifibrotic agent that reduces albuminuria in a rat model of progressive diabetic nephropathy. Bioorg. Med. Chem. Lett. 2013, 23, 6868– 6873, DOI: 10.1016/j.bmcl.2013.09.100[Crossref], [PubMed], [CAS], Google Scholar241https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1yqurrN&md5=2f74b5facaf9c7c186e18ce7bac5dfbb3',4'-Bis-difluoromethoxycinnamoylanthranilate (FT061): An orally-active antifibrotic agent that reduces albuminuria in a rat model of progressive diabetic nephropathyWilliams, Spencer J.; Zammit, Steven C.; Cox, Alison J.; Shackleford, David M.; Morizzi, Julia; Zhang, Yuan; Powell, Andrew K.; Gilbert, Richard E.; Krum, Henry; Kelly, Darren J.Bioorganic & Medicinal Chemistry Letters (2013), 23 (24), 6868-6873CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Cinnamoylanthranilates including tranilast have been identified as promising antifibrotics that can reduce fibrosis occurring in the kidney during diabetes, thereby delaying and/or preventing kidney dysfunction. Structure-activity relationships aimed at improving potency and metabolic stability have led to the discovery of FT061. This compd., which bears a bis-difluoromethoxy catechol, attenuates TGF-β-stimulated prodn. of collagen in cultured renal mesangial cells (approx 50% at 3 μM). When dosed orally at 20 mg/kg to male Sprague Dawley rats, FT061 exhibited a high bioavailability (73%), Cmax of 200 μM and Tmax of 150 min, and a half-life of 5.4 h. FT061 reduced albuminuria when orally dosed in rats at 200 mg kg/day in a late intervention study of a rat model of progressive diabetic nephropathy. The prepn. of cinnamoylanthranilates is also discussed.242Matsson, E. M.; Palm, J. E.; Eriksson, U. G.; Bottner, P.; Lundahl, A.; Knutson, L.; Lennernas, H. Effects of ketoconazole on the in vivo biotransformation and hepatobiliary transport of the thrombin inhibitor AZD0837 in pigs. Drug Metab. Dispos. 2011, 39, 239– 246, DOI: 10.1124/dmd.110.035022[Crossref], [PubMed], [CAS], Google Scholar242https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhslKmsbs%253D&md5=8e320402263368d92309c69a0a20066bEffects of ketoconazole on the in vivo biotransformation and hepatobiliary transport of the thrombin inhibitor AZD0837 in pigsMatsson, Elin M.; Palm, Johan E.; Eriksson, Ulf G.; Bottner, Pernilla; Lundahl, Anna; Knutson, Lars; Lennernaes, HansDrug Metabolism and Disposition (2011), 39 (2), 239-246CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)Ketoconazole has been shown in clin. trials to increase the plasma exposure of the oral anticoagulant prodrug AZD0837 [(2S)-N-{4-[(Z)-amino(methoxyimino)methyl]benzyl}-1-{(2R)-2-[3-chloro-5-(difluoromethoxy)phenyl]-2-hydroxyethanoyl}-azetidine-2-carboxamide] and its active metabolite, AR-H067637 [(2S)-N-{4-[amino(imino)methyl]benzyl}-1-{(2R)-2-[3-chloro-5-(difluoromethoxy)phenyl]-2-hydroxyethanoyl}-azetidine-2-carboxamide]. To investigate the biotransformation of AZD0837 and the effect of ketoconazole on this process, we used an exptl. model in pigs that allows repeated sampling from three blood vessels, the bile duct, and a perfused intestinal segment. The pigs received AZD0837 (500 mg) given enterally either alone (n = 5) or together with single-dose ketoconazole (600 mg) (n = 6). The prodrug (n = 2) and its active metabolite (n = 2) were also administered i.v. to provide ref. doses. The plasma data revealed considerable interindividual variation in the exposure of the prodrug, intermediate metabolite, and active metabolite. However, AR-H067637 was detected at very high concns. in the bile with low variability (Aebile = 53 ± 6% of the enteral dose), showing that the compd. had indeed been formed in all of the animals and efficiently transported into the bile canaliculi. Concomitant dosing with ketoconazole increased the area under the plasma concn.-time curve for AZD0837 (by 99%) and for AR-H067637 (by 51%). The effect on the prodrug most likely arose from inhibited CYP3A-mediated metab. Reduced metab. also seemed to explain the increased plasma exposure of the active compd. because ketoconazole prolonged the terminal half-life with no apparent effect on the extensive biliary excretion and biliary clearance. These in vivo results were supported by in vitro depletion expts. for AR-H067637 in pig liver microsomes with and without the addn. of ketoconazole.243Zhong, D.; Xie, Z.; Chen, X. Metabolism of pantoprazole involving conjugation with glutathione in rats. J. Pharm. Pharmacol. 2005, 57, 341– 349, DOI: 10.1211/0022357055669[Crossref], [PubMed], [CAS], Google Scholar243https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXisFSqurY%253D&md5=4f8e36d0ce177ef9f18a15195b1fe0c5Metabolism of pantoprazole involving conjugation with glutathione in ratsZhong, Dafang; Xie, Zhiyong; Chen, XiaoyanJournal of Pharmacy and Pharmacology (2005), 57 (3), 341-349CODEN: JPPMAB; ISSN:0022-3573. (Pharmaceutical Press)We have investigated the metab. of pantoprazole and have provided an explanation for the formation mechanism of its metabolites. Metabolites found in the urine of rats after oral administration of pantoprazole sodium (25 mg kg-1) were analyzed by liq. chromatog./ion trap mass spectrometry (LC/MSn). The N-acetylcysteine derivs. of benzimidazole (M1) and pyridine (M2), four pyridine-related metabolites (M3-M6), and three benzimidazole-related metabolites (M7-M9) were found, none of which had been reported previously. Five of the metabolites (M1, M2, M3, M7, and M8) were isolated from the urine of rats after oral administration of pantoprazole sodium by semi-preparative HPLC. Structures of these metabolites were identified by a combination anal. of LC/MSn and 1HNMR spectra. Structures of the remaining four metabolites (M4, M5, M6, and M9) were tentatively assigned through LC/MSn. The metabolites M2, M3, M4, M5 and M6 and the other metabolites (M1, M7, M8, and M9) reflected the fate of the pyridine moiety and the benzimidazole moiety, resp. The proposed formation route of M3-M6 was via initial redn. to mercaptopyridine followed by S-methylation, O-demethylation, and S-oxidn. to the corresponding sulfoxide or sulfone. Meanwhile, M8 and M9 were formed via initial redn. to the 5-difluoromethoxy-1H-benzoimidazole-2-thiol (M7) followed by hydroxylation and S-methylation. The metab. of pantoprazole included an attack by glutathione on the benzimidazole-2-carbon and pyridine-7'-carbon. It is an important metabolic pathway of pantoprazole in rats.244Cenacchi, V.; Battaglia, R.; Cinato, F.; Riccardi, B.; Spinabelli, D.; Brogin, G.; Puccini, P.; Pezzetta, D. In vitro and in vivo metabolism of CHF 6001, a selective phosphodiesterase (PDE4) inhibitor. Xenobiotica 2015, 45, 693– 710, DOI: 10.3109/00498254.2015.1014945[Crossref], [PubMed], [CAS], Google Scholar244https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFKhtrbN&md5=800594cece11d6c6f7a830d701183a45In vitro and in vivo metabolism of CHF 6001, a selective phosphodiesterase (PDE4) inhibitorCenacchi, Valentina; Battaglia, Rosangela; Cinato, Flavio; Riccardi, Benedetta; Spinabelli, Daniele; Brogin, Giandomenico; Puccini, Paola; Pezzetta, DanieleXenobiotica (2015), 45 (8), 693-710CODEN: XENOBH; ISSN:0049-8254. (Informa Healthcare)1. The metab. of CHF 6001, a novel PDE4 inhibitor, was detd. in vitro in mouse, rat, dog, monkey and human microsomes and hepatocytes and in vivo in plasma, urine, feces and bile of rats after i.v. and intratracheal administration. 2. The behavior of CHF 6001 in microsomes and hepatocytes changed across species. CYP3A4/5 isoenzymes were identified to be the primary enzymes responsible for the metab. of CHF 6001 in human liver microsomes. 3. In the rat, CHF 6001 was found extensively metabolized in urine, feces and bile, but not in plasma, where CHF 6001 was the main compd. present. The metabolite profiles were different in the four biol. matrixes from both qual. and quant. point of view. 4. CHF 6001 was metabolized through hydrolysis with the formation of the alc. CHF 5956, loss of a chlorine atom, loss of the N-oxide, hydroxylation, loss of the cyclopropylmethyl group in the alc. moiety, conjugation with glucuronic acid, glutathione and cysteine-glycine. 5. The major metabolite present in the bile was isolated and characterized by NMR anal. It derived from CHF 6001 through contraction of the pyridine-N-oxide ring to N-hydroxy pyrrole and conjugation with glucuronic acid.245Boland, S.; Alen, J.; Bourin, A.; Castermans, K.; Boumans, N.; Panitti, L.; Vanormelingen, J.; Leysen, D.; Defert, O. Novel Roflumilast analogs as soft PDE4 inhibitors. Bioorg. Med. Chem. Lett. 2014, 24, 4594– 4597, DOI: 10.1016/j.bmcl.2014.07.016[Crossref], [PubMed], [CAS], Google Scholar245https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVWqs7jM&md5=941e29eda1ba0bb3b2da0bbe14383738Novel Roflumilast analogs as soft PDE4 inhibitorsBoland, Sandro; Alen, Jo; Bourin, Arnaud; Castermans, Karolien; Boumans, Nicki; Panitti, Laura; Vanormelingen, Jessica; Leysen, Dirk; Defert, OlivierBioorganic & Medicinal Chemistry Letters (2014), 24 (18), 4594-4597CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)PDE4 inhibitors are of high interest for treatment of a wide range of inflammatory or autoimmune diseases. Their potential however has not yet been realized due to target-assocd. side effects, resulting in a low therapeutic window. We herein report the design, synthesis and evaluation of novel PDE4 inhibitors contg. a γ-lactone structure. Such mols. are designed to undergo metabolic inactivation when entering circulation, thereby limiting systemic exposure and reducing the risk for side effects. The resulting inhibitors, e.g., I (n = 3, 4), were highly active on both PDE4B1 and PDE4D2 and underwent rapid degrdn. in human plasma by paraoxonase 1. In contrast, their metabolites displayed markedly reduced permeability and/or on-target activity.246Moretto, N.; Caruso, P.; Bosco, R.; Marchini, G.; Pastore, F.; Armani, E.; Amari, G.; Rizzi, A.; Ghidini, E.; De Fanti, R.; Capaldi, C.; Carzaniga, L.; Hirsch, E.; Buccellati, C.; Sala, A.; Carnini, C.; Patacchini, R.; Delcanale, M.; Civelli, M.; Villetti, G.; Facchinetti, F. CHF6001 I: a novel highly potent and selective phosphodiesterase 4 inhibitor with robust anti-inflammatory activity and suitable for topical pulmonary administration. J. Pharmacol. Exp. Ther. 2015, 352, 559– 567, DOI: 10.1124/jpet.114.220541[Crossref], [PubMed], [CAS], Google Scholar246https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXit1Omurw%253D&md5=2be98d624fe49f7aba8673fa8aea8688CHF6001 I: a novel highly potent and selective phosphodiesterase 4 inhibitor with robust anti-inflammatory activity and suitable for topical pulmonary administrationMoretto, Nadia; Caruso, Paola; Bosco, Raffaella; Marchini, Gessica; Pastore, Fiorella; Armani, Elisabetta; Amari, Gabriele; Rizzi, Andrea; Ghidini, Eleonora; De Fanti, Renato; Capaldi, Carmelida; Carzaniga, Laura; Hirsch, Emilio; Buccellati, Carola; Sala, Angelo; Carnini, Chiara; Patacchini, Riccardo; Delcanale, Maurizio; Civelli, Maurizio; Villetti, Gino; Facchinetti, FabrizioJournal of Pharmacology and Experimental Therapeutics (2015), 352 (3), 559-567, 9 pp.CODEN: JPETAB; ISSN:1521-0103. (American Society for Pharmacology and Experimental Therapeutics)This study examd. the pharmacol. characterization of CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide], a novel phosphodiesterase (PDE)4 inhibitor designed for treating pulmonary inflammatory diseases via inhaled administration. CHF6001 was 7- and 923-fold more potent than roflumilast and cilomilast, resp., in inhibiting PDE4 enzymic activity (IC50 = 0.026 ± 0.006 nM). CHF6001 inhibited PDE4 isoforms A-D with equal potency, showed an elevated ratio of high-affinity rolipram binding site vs. low-affinity rolipram binding site (i.e., >40) and displayed >20,000-fold selectivity vs. PDE4 compared with a panel of PDEs. CHF6001 effectively inhibited (subnanomolar IC50 values) the release of tumor necrosis factor-α from human peripheral blood mononuclear cells, human acute monocytic leukemia cell line macrophages (THP-1), and rodent macrophages (RAW264.7 and NR8383). Moreover, CHF6001 potently inhibited the activation of oxidative burst in neutrophils and eosinophils, neutrophil chemotaxis, and the release of interferon-γ from CD4+ T cells. In all these functional assays, CHF6001 was more potent than previously described PDE4 inhibitors, including roflumilast, UK-500,001 [2-(3,4-difluorophenoxy)-5-fluoro-N-((1S,4S)-4-(2-hydroxy-5-methylbenzamido)cyclohexyl)nicotinamide], and cilomilast, and it was comparable to GSK256066 [6-((3-(dimethylcarbamoyl)phenyl)sulfonyl)-4-((3-methoxyphenyl)amino)-8-methylquinoline-3-carboxamide]. When administered intratracheally to rats as a micronized dry powder, CHF6001 inhibited liposaccharide-induced pulmonary neutrophilia (ED50 = 0.205 μmol/kg) and leukocyte infiltration (ED50 = 0.188 μmol/kg) with an efficacy comparable to a high dose of budesonide (1 μmol/kg i.p.). In sum, CHF6001 has the potential to be an effective topical treatment of conditions assocd. with pulmonary inflammation, including asthma and chronic obstructive pulmonary disease.247Zhou, Z. Z.; Ge, B. C.; Zhong, Q. P.; Huang, C.; Cheng, Y. F.; Yang, X. M.; Wang, H. T.; Xu, J. P. Development of highly potent phosphodiesterase 4 inhibitors with anti-neuroinflammation potential: Design, synthesis, and structure-activity relationship study of catecholamides bearing aromatic rings. Eur. J. Med. Chem. 2016, 124, 372– 379, DOI: 10.1016/j.ejmech.2016.08.052[Crossref], [PubMed], [CAS], Google Scholar247https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFWisLzL&md5=a94bd083cfeb43381366a854a1624516Development of highly potent phosphodiesterase 4 inhibitors with anti-neuroinflammation potential: Design, synthesis, and structure-activity relationship study of catecholamides bearing aromatic ringsZhou, Zhong-Zhen; Ge, Bing-Chen; Zhong, Qiu-Ping; Huang, Chang; Cheng, Yu-Fang; Yang, Xue-Mei; Wang, Hai-Tao; Xu, Jiang-PingEuropean Journal of Medicinal Chemistry (2016), 124 (), 372-379CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)In this study, catecholamides I (R=Me,CHF2;R1=cyclopentyl, cyclopropylmethyl; R2=H,Me; R3=H,Cl;X=Y=N,C) bearing different arom. rings (such as pyridin-2-yl, pyridin-3-yl,Ph,and 2-chlorophenyl groups) were synthesized as potent phosphodiesterase(PDE)4 inhibitors. The inhibitory activities of these compds. were evaluated against the core catalytic domains of human PDE4(PDE4CAT), full-length PDE4A4, PDE4B1, PDE4C1, and PDE4D7 enzymes, and other PDE family members. Eight of the synthesized compds. were identified as having submicromolar IC50 values in the mid-to low-nanomolar range. Careful anal. on the structure-activity relationship of compds. I revealed that the replacement of the 4-methoxy group with the difluoromethoxy group improved inhibitory activities. More interesting, 4-difluoromethoxybenzamides I (R=CHF2; R1=cyclopentyl, cyclopropylmethyl; R2=H; R3=Cl; X=Y=C) exhibited preference for PDE4 with higher selectivities of about 3333 and 1111-fold over other PDEs, resp. In addn., compd. I (R=CHF2; R1=cyclopropylmethyl; R2=H; R3=Cl; X=Y=C) with wonderful PDE4D7 inhibitory activities inhibited LPS-induced TNF-α prodn. in microglia.248Brullo, C.; Massa, M.; Villa, C.; Ricciarelli, R.; Rivera, D.; Pronzato, M. A.; Fedele, E.; Barocelli, E.; Bertoni, S.; Flammini, L.; Bruno, O. Synthesis, biological activities and pharmacokinetic properties of new fluorinated derivatives of selective PDE4D inhibitors. Bioorg. Med. Chem. 2015, 23, 3426– 3435, DOI: 10.1016/j.bmc.2015.04.027[Crossref], [PubMed], [CAS], Google Scholar248https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmslGisL0%253D&md5=be893b649c0211a01e2b4a15fa14a999Synthesis, biological activities and pharmacokinetic properties of new fluorinated derivatives of selective PDE4D inhibitorsBrullo, Chiara; Massa, Matteo; Villa, Carla; Ricciarelli, Roberta; Rivera, Daniela; Pronzato, Maria Adelaide; Fedele, Ernesto; Barocelli, Elisabetta; Bertoni, Simona; Flammini, Lisa; Bruno, OlgaBioorganic & Medicinal Chemistry (2015), 23 (13), 3426-3435CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)A new series of selective PDE4D inhibitors has been designed and synthesized by replacing 3-methoxy group with 3-difluoromethoxy isoster moiety in our previously reported cathecolic structures. All compds. showed a good PDE4D3 inhibitory activity, most of them being inactive toward other PDE4 isoforms (PDE4A4, PDE4B2 and PDE4C2). Compd. 3b, chosen among the synthesized compds. as the most promising in terms of inhibitory activity, selectivity and safety, showed an improved pharmacokinetic profile compared to its non fluorinated analog. Spontaneous locomotor activity, assessed in an open field app., showed that, differently from rolipram and diazepam, selective PDE4D inhibitors, such as compds. 3b, 5b and 7b, did not affect locomotion, whereas compd. 1b showed a tendency to reduce the distance traveled and to prolong the immobility period, possibly due to a poor selectivity.249Guay, D.; Hamel, P.; Blouin, M.; Brideau, C.; Chan, C. C.; Chauret, N.; Ducharme, Y.; Huang, Z.; Girard, M.; Jones, T. R.; Laliberte, F.; Masson, P.; McAuliffe, M.; Piechuta, H.; Silva, J.; Young, R. N.; Girard, Y. Discovery of L-791,943: a potent, selective, non emetic and orally active phosphodiesterase-4 inhibitor. Bioorg. Med. Chem. Lett. 2002, 12, 1457– 1461, DOI: 10.1016/S0960-894X(02)00190-7[Crossref], [PubMed], [CAS], Google Scholar249https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xjsl2lsbY%253D&md5=d52b627cf615fa8a862625ae001c8181Discovery of L-791,943: A potent, selective, non emetic and orally active phosphodiesterase-4 inhibitorGuay, Daniel; Hamel, Pierre; Blouin, Marc; Brideau, Christine; Chan, Chi Chung; Chauret, Nathalie; Ducharme, Yves; Huang, Zheng; Girard, Mario; Jones, Tom R.; Laliberte, France; Masson, Paul; McAuliffe, Malia; Piechuta, Hanna; Silva, Jose; Young, Robert N.; Girard, YvesBioorganic & Medicinal Chemistry Letters (2002), 12 (11), 1457-1461CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science Ltd.)Structure-activity relationship studies directed toward improving the potency and metabolic stability of CDP-840 resulted in the discovery of L-791,943 as a potent (HWB TNF-α=0.67 μM) and orally active phosphodiesterase type 4 (PDE4) inhibitor. This compd., which bears a stable bis-difluoromethoxy catechol and a pendant hexafluorocarbinol, exhibited a long half-life in rat and in squirrel monkey. It is well tolerated in ferret with an emetic threshold greater than 30 mg/kg (po) and was found to be active in the ovalbumin-induced bronchoconstriction model in guinea pig and in the ascaris-induced bronchoconstriction models in sheep and squirrel monkey.250Zhuo, X.; Hartz, R. A.; Bronson, J. J.; Wong, H.; Ahuja, V. T.; Vrudhula, V. M.; Leet, J. E.; Huang, S.; Macor, J. E.; Shu, Y. Z. Comparative biotransformation of pyrazinone-containing corticotropin-releasing factor receptor-1 antagonists: minimizing the reactive metabolite formation. Drug Metab. Dispos. 2010, 38, 5– 15, DOI: 10.1124/dmd.109.028910[Crossref], [PubMed], [CAS], Google Scholar250https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhs1aru7fE&md5=ce4d9c5f99f96cbc018968130b068e81Comparative biotransformation of pyrazinone-containing corticotropin-releasing factor receptor-1 antagonists: minimizing the reactive metabolite formationZhuo, Xiaoliang; Hartz, Richard A.; Bronson, Joanne J.; Wong, Harvey; Ahuja, Vijay T.; Vrudhula, Vivekananda M.; Leet, John E.; Huang, Stella; Macor, John E.; Shu, Yue-ZhongDrug Metabolism and Disposition (2010), 38 (1), 5-15CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)(S)-5-Chloro-1-(1-cyclopropylethyl)-3-(2,6-dichloro-4-(trifluoromethyl)-phenylamino)pyrazin-2(1H)-one (BMS-665053), a pyrazinone-contg. compd., is a potent and selective antagonist of corticotropin-releasing factor receptor-1 (CRF-R1) that showed efficacy in the defensive withdrawal model for anxiety in rats, suggesting its use as a potential treatment for anxiety and depression. In vitro metab. studies of BMS-665053 in rat and human liver microsomes revealed cytochrome P 450-mediated oxidn. of the pyrazinone moiety, followed by ring opening, as the primary metabolic pathway. Detection of a series of GSH adducts in trapping expts. suggested the formation of a reactive intermediate, probably as a result of epoxidn. of the pyrazinone moiety. In addn., BMS-665053 (20 mg/kg i.v.) underwent extensive metab. in bile duct-cannulated (BDC) rats. The major drug-related materials in rat plasma were the pyrazinone oxidn. products. In rat bile and urine (0-7 h), only a trace amt. of the parent drug was recovered, whereas significant levels of the pyrazinone epoxide-derived metabolites and GSH-related conjugates were detected. Further evidence suggested that GSH-related conjugates also formed at the dichloroarylamine moiety possibly via an epoxide or a quinone imine intermediate. Other major metabolites in BDC rat bile and urine included glucuronide conjugates. To reduce potential liability due to metabolic activation of BMS-665053, a no. of pyrazinone analogs with different substituents were synthesized and investigated for reactive metabolite formation, leading to the discovery of a CRF-R1 antagonist with diminished in vitro metabolic activation.251Olah, G. A.; Nojima, M.; Kerekes, I. Synthetic methods and reactions. IV. Fluorination of carboxylic acids with cyanuric fluoride. Synthesis 1973, 1973, 487– 488, DOI: 10.1055/s-1973-22238252Wei, C.; Chupak, L. S.; Philip, T.; Johnson, B. M.; Gentles, R.; Drexler, D. M. Screening and characterization of reactive compounds with in vitro peptide-trapping and liquid chromatography/high-resolution accurate mass spectrometry. J. Biomol. Screening 2014, 19, 297– 307, DOI: 10.1177/1087057113492852[Crossref], [PubMed], [CAS], Google Scholar252https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhslWgs7vN&md5=49f09c44ffdd887b3523575a8af4d838Screening and characterization of reactive compounds with in vitro peptide-trapping and liquid chromatography/high-resolution accurate mass spectrometryWei, Cong; Chupak, Louis S.; Philip, Thomas; Johnson, Benjamin M.; Gentles, Robert; Drexler, Dieter M.Journal of Biomolecular Screening (2014), 19 (2), 297-307, 11CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)The present study describes a novel methodol. for the detection of reactive compds. using in vitro peptide trapping and liq. chromatog.-high-resoln. accurate mass spectrometry (LC-HRMS). Compds. that contain electrophilic groups can covalently bind to nucleophilic moieties in proteins and form adducts. Such adducts are thought to be assocd. with drug-mediated toxicity and therefore represent potential liabilities in drug discovery programs. In addn., reactive compds. identified in biol. screening can be assocd. with data that can be misinterpreted if the reactive nature of the compd. is not appreciated. In this work, to facilitate the triage of hits from high-throughput screening (HTS), a novel assay was developed to monitor the formation of covalent peptide adducts by compds. suspected to be chem. reactive. The assay consists of in vitro incubations of test compds. (under conditions of physiol. pH) with synthetically prepd. peptides presenting a variety of nucleophilic moieties such as cysteine, lysine, histidine, arginine, serine, and tyrosine. Reaction mixts. were analyzed using full-scan LC-HRMS, the data were interrogated using postacquisition data mining, and modified amino acids were identified by subsequent LC-HRMS/mass spectrometry. The study demonstrated that in vitro nucleophilic peptide trapping followed by LC-HRMS anal. is a useful approach for screening of intrinsically reactive compds. identified from HTS exercises, which are then removed from follow-up processes, thus obviating the generation of data from biochem. activity assays.253Rodil, A.; Slawin, A. M. Z.; Al-Maharik, N.; Tomita, R.; O’Hagan, D. O. Fluorine-containing substituents: metabolism of the α,α-difluoroethyl thioether motif. Beilstein J. Org. Chem. 2019, 15, 1441– 1447, DOI: 10.3762/bjoc.15.144[Crossref], [PubMed], [CAS], Google Scholar253https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1eitbvJ&md5=967d18a4e9cf6c8f3935a4a7e19065d0Fluorine-containing substituents: metabolism of the α,α-difluoroethyl thioether motifRodil, Andrea; Slawin, Alexandra M. Z.; Al-Maharik, Nawaf; Tomita, Ren; O'Hagan, DavidBeilstein Journal of Organic Chemistry (2019), 15 (), 1441-1447CODEN: BJOCBH; ISSN:1860-5397. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)We report the metab. of the recently introduced α,α-difluoroethyl thioether motif to explore further its potential as a substituent for bioactives discovery chem. Incubation of two aryl-SCF2CH3 ethers with the model yeast organism Cunninghamella elegans, indicates that the sulfur of the thioether is rapidly converted to the corresponding sulfoxide, and then significantly more slowly to the sulfone. When the substrate was (p-OMe)PhSCF2CH3, then the resultant (demethylated) phenol sulfoxide had an enantiomeric excess of 60%, and when the substrate was the β-substituted-SCF2CH3 naphthalene, then the enantiomeric excess of the resultant sulfoxide was 54%. There was no evidence of defluorination, unlike the corresponding oxygen ether (p-OMe)PhOCF2CH3, which was converted to the (demethylated) phenol acetate ester during C. elegans incubation. We conclude that the aryl-S-CF2CH3 motif is metabolised in a similar manner to aryl-SCF3, a motif that is being widely explored in discovery chem. It is however, significantly less lipophilic than aryl-SCF3 which may offer a practical advantage in tuning overall pharmacokinetic profiles of mols. in development.254Tomita, R.; Al-Maharik, N.; Rodil, A.; Buhl, M.; O’Hagan, D. Synthesis of aryl α,α-difluoroethyl thioethers a novel structure motif in organic chemistry, and extending to aryl α,α-difluoro oxyethers. Org. Biomol. Chem. 2018, 16, 1113– 1117, DOI: 10.1039/C7OB02987J[Crossref], [PubMed], [CAS], Google Scholar254https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXosVKmsw%253D%253D&md5=c8144df20378eac6ac827338da2bba94Synthesis of aryl α,α-difluoroethyl thioethers a novel structure motif in organic chemistry, and extending to aryl α,α-difluoro oxyethersTomita, Ren; Al-Maharik, Nawaf; Rodil, Andrea; Buhl, Michael; O'Hagan, DavidOrganic & Biomolecular Chemistry (2018), 16 (7), 1113-1117CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)A method for the prepn. of aryl α,α-difluoroethyl thioethers ArSCF2CH3 (Ar = C6H5, naphthalen-2-yl, 4-(cyclohexyloxy)phenyl, etc.) is reported and the synthesis approach is extended to aryl α,α-difluoroethyl oxygen ethers 4-RC6H4OCF2CH3 (R = H, OCH3, C6H5). Selected building blocks are further elaborated in cross-coupling reactions and are incorporated into analogs of established trifluoromethyl ether drugs. Conformations are explored and log P studies of these motifs indicate that they are significantly more polar than their trifluoromethyl ether analogs rendering them attractive for bioactives discovery.255Murray, M. Mechanisms of inhibitory and regulatory effects of methylenedioxyphenyl compounds on cytochrome P450-dependent drug oxidation. Curr. Drug Metab. 2000, 1, 67– 84, DOI: 10.2174/1389200003339270[Crossref], [PubMed], [CAS], Google Scholar255https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXlt1Whurs%253D&md5=dcafc824c8bcd0080d256d6dd8c50ae7Mechanisms of inhibitory and regulatory effects of methylenedioxyphenyl compounds on cytochrome P450-dependent drug oxidationMurray, MichaelCurrent Drug Metabolism (2000), 1 (1), 67-84CODEN: CDMUBU ISSN:. (Bentham Science Publishers Ltd.)A review with 110 refs. Cytochrome P 450 (CYP) enzymes catalyze the oxidative conversion of drugs and other lipophilic compds. to hydrophilic metabolites. Thus, CYPs play a dominant role in the elimination of drugs from the body. Inhibitory interactions occur when drugs compete for oxidn. by specific CYPs, whereas certain drugs increase the capacity for oxidative biotransformation by inducing the synthesis of new CYPs. Methylenedioxyphenyl (MDP) compds. have been widely employed as com. important pesticide synergists and a no. of derivs. are found in oils and spices. MDP compds. are of considerable toxicol. significance because of their capacity to inhibit and induce CYP enzymes in mammals; some derivs. produce neurotoxic and hepatotoxic effects. Although there are relatively few therapeutic agents of present clin. importance that possess the MDP structural feature, the synthesis and preclin. evaluation of such agents appears to be increasing. In the context of the existing literature surrounding MDP compds. it is noteworthy that these potential drugs also elicit significant modulatory effects on CYP activities in rat and human liver. These developments indicate the importance of understanding the chem. mechanisms by which MDPs interact with CYPs. Thus, the presence of the MDP structure may undermine the potential clin. value of new drugs.256Bartholomaeus, A. Fludioxonil. In World Health Organization Joint Meeting on Pesticide Residues; World Health Organization, 2004; pp 47– 84.257Keith, J. M.; Jones, W. M.; Tichenor, M.; Liu, J.; Seierstad, M.; Palmer, J. A.; Webb, M.; Karbarz, M.; Scott, B. P.; Wilson, S. J.; Luo, L.; Wennerholm, M. L.; Chang, L.; Rizzolio, M.; Rynberg, R.; Chaplan, S. R.; Breitenbucher, J. G. Preclinical characterization of the FAAH inhibitor JNJ-42165279. ACS Med. Chem. Lett. 2015, 6, 1204– 1208, DOI: 10.1021/acsmedchemlett.5b00353[ACS Full Text], [CAS], Google Scholar257https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhslGms73P&md5=fc681414ace29f39906fdd62c204114bPreclinical Characterization of the FAAH Inhibitor JNJ-42165279Keith, John M.; Jones, William M.; Tichenor, Mark; Liu, Jing; Seierstad, Mark; Palmer, James A.; Webb, Michael; Karbarz, Mark; Scott, Brian P.; Wilson, Sandy J.; Luo, Lin; Wennerholm, Michelle L.; Chang, Leon; Rizzolio, Michele; Rynberg, Raymond; Chaplan, Sandra R.; Breitenbucher, J. GuyACS Medicinal Chemistry Letters (2015), 6 (12), 1204-1208CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)The preclin. characterization of the aryl piperazinyl urea inhibitor of fatty acid amide hydrolase (FAAH) JNJ-42165279 I is described. JNJ-42165279 covalently inactivates the FAAH enzyme, but is highly selective with regard to other enzymes, ion channels, transporters, and receptors. JNJ-42165279 exhibited excellent ADME and pharmacodynamic properties as evidenced by its ability to block FAAH in the brain and periphery of rats and thereby cause an elevation of the concns. of anandamide (AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The compd. was also efficacious in the spinal nerve ligation (SNL) model of neuropathic pain. The combination of good phys., ADME, and PD properties of JNJ-42165279 supported it entering the clin. portfolio.258Rose, W. C.; Marathe, P. H.; Jang, G. R.; Monticello, T. M.; Balasubramanian, B. N.; Long, B.; Fairchild, C. R.; Wall, M. E.; Wani, M. C. Novel fluoro-substituted camptothecins: in vivo antitumor activity, reduced gastrointestinal toxicity and pharmacokinetic characterization. Cancer Chemother. Pharmacol. 2006, 58, 73– 85, DOI: 10.1007/s00280-005-0128-y[Crossref], [PubMed], [CAS], Google Scholar258https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjslGjsbg%253D&md5=97ecbd7d19b5e051255c5c0dc03b215aNovel fluoro-substituted camptothecins: in vivo antitumor activity, reduced gastrointestinal toxicity and pharmacokinetic characterizationRose, William C.; Marathe, Punit H.; Jang, Graham R.; Monticello, Thomas M.; Balasubramanian, Balu N.; Long, Byron; Fairchild, Craig R.; Wall, Monroe E.; Wani, Mansukh C.Cancer Chemotherapy and Pharmacology (2006), 58 (1), 73-85CODEN: CCPHDZ; ISSN:0344-5704. (Springer)Purpose: The novel fluoro-substituted camptothecin analog, BMS-286309, and its prodrug, BMS-422461, were evaluated for their pharmacol., toxicol., metabolic and pharmacokinetic developmental potential. Methods: In vitro and in vivo assays were used to assess the compds. for topoisomerase I activity, antitumor activity, gastrointestinal (GI) toxicity, and pharmacokinetic parameters. Results: BMS-286309-induced topoisomerase I-mediated DNA breaks in vitro and was similar in potency to camptothecin. Both BMS-286309 and -422461 were comparable to irinotecan regarding preclin. antitumor activity assessed in mice bearing distal site murine and human tumors. BMS-422461 was also found to be orally active. Both analogs were >100-fold more potent in vivo than irinotecan and both were superior to irinotecan with respect to toxicol. assessment of GI injury in mice. The generation of parent compd. from BMS-422461 was qual. similar in mouse, rat and human blood and liver S9 fractions. The percentage of BMS-286309 remaining as the active lactone form at equil. was comparable in mouse and human plasma. The pharmacokinetic profile in rat blood demonstrated that BMS-422461 was rapidly cleaved to BMS-286309. Conclusions: The favorable in vivo metabolic activation of BMS-422461, and the pharmacokinetic characteristics of BMS-286309, suggest that the good efficacy of BMS-422461 is derived from robust in vivo release of BMS-286309 in rodents and the likelihood that this biotransformation will be preserved in humans. The comparable antitumor activity of BMS-422461 to irinotecan, as well as reduced preclin. GI toxicity, make this novel camptothecin analog attractive for clin. development.259Perfetti, X.; O’Mathuna, B.; Pizarro, N.; Cuyas, E.; Khymenets, O.; Almeida, B.; Pellegrini, M.; Pichini, S.; Lau, S. S.; Monks, T. J.; Farre, M.; Pascual, J. A.; Joglar, J.; de la Torre, R. Neurotoxic thioether adducts of 3,4-methylenedioxymethamphetamine identified in human urine after ecstasy ingestion. Drug Metab. Dispos. 2009, 37, 1448– 1455, DOI: 10.1124/dmd.108.026393[Crossref], [PubMed], [CAS], Google Scholar259https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXosFemsrs%253D&md5=e32e277a1b10c6423f624afecca89f6fNeurotoxic thioether adducts of 3,4-methylenedioxymethamphetamine identified in human urine after ecstasy ingestionPerfetti, Ximena; O'Mathuna, Brian; Pizarro, Nieves; Cuyas, Elisabet; Khymenets, Olha; Almeida, Bruno; Pellegrini, Manuela; Pichini, Simona; Lau, Serrine S.; Monks, Terrence J.; Farre, Magi; Pascual, Jose Antonio; Joglar, Jesus; de la Torre, RafaelDrug Metabolism and Disposition (2009), 37 (7), 1448-1455CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) is a widely misused synthetic amphetamine deriv. and a serotonergic neurotoxicant in animal models and possibly humans. The underlying mechanism of neurotoxicity involves the formation of reactive oxygen species although their source remains unclear. It has been postulated that MDMA-induced neurotoxicity is mediated via the formation of bioreactive metabolites. In particular, the primary catechol metabolites, 3,4-dihydroxymethamphetamine (HHMA) and 3,4-dihydroxyamphetamine (HHA), subsequently cause the formation of glutathione and N-acetylcysteine conjugates, which retain the ability to redox cycle and are serotonergic neurotoxicants in rats. Although the presence of such metabolites has been recently demonstrated in rat brain microdialyzate, their formation in humans has not been reported. The present study describes the detection of 5-(N-acetylcystein-S-yl)-3,4-dihydroxymethamphetamine (N-Ac-5-Cys-HHMA) and 5-(N-acetylcystein-S-yl)-3,4-dihydroxyamphetamine (N-Ac-5-Cys-HHA) in human urine of 15 recreational users of MDMA (1.5 mg/kg) in a controlled setting. The results reveal that in the first 4 h after MDMA ingestion ∼0.002% of the administered dose was recovered as thioether adducts. Genetic polymorphisms in CYP2D6 and catechol-O-methyltransferase expression, the combination of which are major determinants of steady-state levels of HHMA and 4-hydroxy-3-methoxyamphetamine, probably explain the interindividual variability seen in the recovery of N-Ac-5-Cys-HHMA and N-Ac-5-Cys-HHA. In summary, the formation of neurotoxic thioether adducts of MDMA has been demonstrated for the first time in humans. The findings lend wt. to the hypothesis that the bioactivation of MDMA to neurotoxic metabolites is a relevant pathway to neurotoxicity in humans.260Ot’alora G, M.; Grigsby, J.; Poulter, B.; Van Derveer, J. W., III; Giron, S. G.; Jerome, L.; Feduccia, A. A.; Hamilton, S.; Yazar-Klosinski, B.; Emerson, A.; Mithoefer, M. C.; Doblin, R. 3,4-Methylenedioxymethamphetamine-assisted psychotherapy for treatment of chronic posttraumatic stress disorder: A randomized phase 2 controlled trial. J. Psychopharmacol. 2018, 32, 1295– 1307, DOI: 10.1177/0269881118806297[Crossref], [PubMed], [CAS], Google Scholar260https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXit1ymur%252FL&md5=a3cff037100d9034378f62498b4e7eba3,4-Methylenedioxymethamphetamine-assisted psychotherapy for treatment of chronic posttraumatic stress disorder: A randomized phase 2 controlled trialOt'alora G, Marcela; Grigsby, Jim; Poulter, Bruce; Van Derveer, Joseph W.; Giron, Sara Gael; Jerome, Lisa; Feduccia, Allison A.; Hamilton, Scott; Yazar-Klosinski, Berra; Emerson, Amy; Mithoefer, Michael C.; Doblin, RickJournal of Psychopharmacology (London, United Kingdom) (2018), 32 (12), 1295-1307CODEN: JOPSEQ; ISSN:0269-8811. (Sage Publications Ltd.)Posttraumatic stress disorder often does not resolve after conventional psychotherapies or pharmacotherapies. Pilot studies have reported that 3,4-methylenedioxymethamphetamine (MDMA) combined with psychotherapy reduces posttraumatic stress disorder symptoms. This pilot dose response trial assessed efficacy and safety of MDMA-assisted psychotherapy across multiple therapy teams. Twenty-eight people with chronic posttraumatic stress disorder were randomized in a double-blind dose response comparison of two active doses (100 and 125 mg) with a low dose (40 mg) of MDMA administered during eight-hour psychotherapy sessions. Change in the Clinician-Administered PTSD Scale total scores one month after two sessions of MDMA served as the primary outcome. Active dose groups had one addnl. open-label session; the low dose group crossed over for three open-label active dose sessions. A 12-mo follow-up assessment occurred after the final MDMA session. In the intent-to-treat set, the active groups had the largest redn. in Clinician-Administered PTSD Scale total scores at the primary endpoint, with mean (std. deviation) changes of -26.3 (29.5) for 125 mg, -24.4 (24.2) for 100 mg, and -11.5 (21.2) for 40 mg, though statistical significance was reached only in the per protocol set (p = 0.03). Posttraumatic stress disorder symptoms remained lower than baseline at 12-mo follow-up (p<0.001) with 76% (n = 25) not meeting posttraumatic stress disorder criteria. There were no drug-related serious adverse events, and the treatment was well-tolerated. Our findings support previous investigations of MDMA-assisted psychotherapy as an innovative, efficacious treatment for posttraumatic stress disorder.261Ricaurte, G. A.; Yuan, J.; Hatzidimitriou, G.; Cord, B. J.; McCann, U. D. Retraction. Science 2003, 301, 1479, DOI: 10.1126/science.301.5639.1479b[Crossref], [PubMed], [CAS], Google Scholar261https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXnt1Kqtr4%253D&md5=2d714a34872298db242f49dd3003c49cRetractionRicaurte, George A.; Yuan, Jie; Hatzidmitriou, George; Cord, Branden J.; McCann, Una D.Science (Washington, DC, United States) (2003), 301 (5639), 1479CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)There is no expanded citation for this reference.262Center for Drug Evaluation and Research. Application Number: 206038Orig1s000. Clinical Pharmacology and Biopharmaceutics Review(s). https://www.accessdata.fda.gov/drugsatfda_docs/nda/2015/0206038Orig1s000ClinPharmR.pdf (accessed 2019-05-06).263Bertelsen, K. M.; Venkatakrishnan, K.; Von Moltke, L. L.; Obach, R. S.; Greenblatt, D. J. Apparent mechanism-based inhibition of human CYP2D6 in vitro by paroxetine: comparison with fluoxetine and quinidine. Drug Metab. Dispos. 2003, 31, 289– 293, DOI: 10.1124/dmd.31.3.289[Crossref], [PubMed], [CAS], Google Scholar263https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhsFKlt7Y%253D&md5=2336fbc058e49e7b530562809c62ba09Apparent mechanism-based inhibition of human CYP2D6 in vitro by paroxetine: Comparison with fluoxetine and quinidineBertelsen, Kirk M.; Venkatakrishnan, Karthik; Von Moltke, Lisa L.; Obach, R. Scott; Greenblatt, David J.Drug Metabolism and Disposition (2003), 31 (3), 289-293CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)Paroxetine, a selective serotonin reuptake inhibitor, is a potent inhibitor of cytochrome P 450 2D6 (CYP2D6) activity, but the mechanism of inhibition is not established. To det. whether preincubation affects the inhibition of human liver microsomal dextromethorphan demethylation activity by paroxetine, we used a two-step incubation scheme in which all of the enzyme assay components, minus substrate, are preincubated with paroxetine. The kinetic parameters of inhibition were also estd. by varying the time of preincubation as well as the concn. of inhibitor. From these data, a Kitz-Wilson plot was constructed, allowing the estn. of both an apparent inactivator concn. required for half-maximal inactivation (KI) and the maximal rate const. of inactivation (KINACT) value for this interaction. Preincubation of paroxetine with human liver microsomes caused an approx. 8-fold redn. in the IC50 value (0.34 vs. 2.54 μM). Time-dependent inhibition was demonstrated with an apparent KI of 4.85 μM and an apparent KINACT value of 0.17 min-1. Spectral scanning of CYP2D6 with paroxetine yielded an increase in absorbance at 456 nm suggesting paroxetine inactivation of CYP2D6 via the formation of a metabolite intermediate complex. This pattern is consistent with the metab. of the methylenedioxy substituent in paroxetine; such substituents may produce mechanism-based inactivation of cytochrome P 450 enzymes. In contrast, quinidine and fluoxetine, both of which are inhibitors of CYP2D6 activity, did not exhibit a preincubation-dependent increase in inhibitory potency. These data are consistent with mechanism-based inhibition of CYP2D6 by paroxetine but not by quinidine or fluoxetine.264Halazy, S.; Danzin, C.; Ehrhard, A.; Gerhart, F. 1,1-Difluoroalkyl glucosides: a new class of enzyme-activated irreversible inhibitors of α-glucosidases. J. Am. Chem. Soc. 1989, 111, 3484– 3485, DOI: 10.1021/ja00191a085[ACS Full Text], [CAS], Google Scholar264https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXhvFehtrs%253D&md5=8f71ee79807e7ee4cd2713e3c338ce681,1-Difluoroalkyl glucosides: a new class of enzyme-activated irreversible inhibitors of α-glucosidasesHalazy, S.; Danzin, C.; Ehrhard, A.; Gerhart, F.Journal of the American Chemical Society (1989), 111 (9), 3484-5CODEN: JACSAT; ISSN:0002-7863.2'-Chloro-1',1',2'-trifluoroethyl α-D-glucopyranoside (I) was synthesized as a potential inhibitor of α-glucosidase. I produced a time-dependent irreversible inhibition of yeast α-glucosidase. The inactivation is explained by the enzyme-catalyzed hydrolysis of the glycosidic linkage releasing a fluorohydrin which rapidly decomps. to an acyl fluoride and acetylates a nucleophilic residue in the active site. I was found to be a substrate of the sucrase-isomaltase complex purified from rat small intestine.265Vocadlo, D. J.; Davies, G. J. Mechanistic insights into glycosidase chemistry. Curr. Opin. Chem. Biol. 2008, 12, 539– 555, DOI: 10.1016/j.cbpa.2008.05.010[Crossref], [PubMed], [CAS], Google Scholar265https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXht1KgsbnF&md5=dc269026d21f0e114c25b120b3c1e9b9Mechanistic insights into glycosidase chemistryVocadlo, David J.; Davies, Gideon J.Current Opinion in Chemical Biology (2008), 12 (5), 539-555CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. The enzymic hydrolysis of the glycosidic bond continues to gain importance, reflecting the critically important roles complex glycans play in health and disease as well as the rekindled interest in enzymic biomass conversion. Recent advances include the broadening of our understanding of enzyme reaction coordinates, through both computational and structural studies, improved understanding of enzyme inhibition through transition state mimicry and fascinating insights into mechanism yielded by phys. org. chem. approaches.266Spahn, V.; Del Vecchio, G.; Labuz, D.; Rodriguez-Gaztelumendi, A.; Massaly, N.; Temp, J.; Durmaz, V.; Sabri, P.; Reidelbach, M.; Machelska, H.; Weber, M.; Stein, C. A nontoxic pain killer designed by modeling of pathological receptor conformations. Science 2017, 355, 966– 969, DOI: 10.1126/science.aai8636[Crossref], [PubMed], [CAS], Google Scholar266https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjsVCgs7k%253D&md5=719809495aec8c3ac9e60429c9edea98A nontoxic pain killer designed by modeling of pathological receptor conformationsSpahn, V.; Del Vecchio, G.; Labuz, D.; Rodriguez-Gaztelumendi, A.; Massaly, N.; Temp, J.; Durmaz, V.; Sabri, P.; Reidelbach, M.; Machelska, H.; Weber, M.; Stein, C.Science (Washington, DC, United States) (2017), 355 (6328), 966-969CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Indiscriminate activation of opioid receptors provides pain relief but also severe central and intestinal side effects. We hypothesized that exploiting pathol. (rather than physiol.) conformation dynamics of opioid receptor-ligand interactions might yield ligands without adverse actions. By computer simulations at low pH, a hallmark of injured tissue, we designed an agonist that, because of its low acid dissocn. const., selectively activates peripheral μ-opioid receptors at the source of pain generation. Unlike the conventional opioid fentanyl, this agonist showed pH-sensitive binding, heterotrimeric guanine nucleotide-binding protein (G protein) subunit dissocn. by fluorescence resonance energy transfer, and adenosine 3',5'-monophosphate inhibition in vitro. It produced injury-restricted analgesia in rats with different types of inflammatory pain without exhibiting respiratory depression, sedation, constipation, or addiction potential.267Grunewald, G. L.; Seim, M. R.; Lu, J.; Makboul, M.; Criscione, K. R. Application of the Goldilocks effect to the design of potent and selective inhibitors of phenylethanolamine N-methyltransferase: balancing pKa and steric effects in the optimization of 3-methyl-1,2,3,4-tetrahydroisoquinoline inhibitors by β-fluorination. J. Med. Chem. 2006, 49, 2939– 2952, DOI: 10.1021/jm051262k[ACS Full Text], [CAS], Google Scholar267https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjsVajtb4%253D&md5=468ae2a5832e69d751628ca743ef951cApplication of the Goldilocks Effect to the Design of Potent and Selective Inhibitors of Phenylethanolamine N-Methyltransferase: Balancing pKa and Steric Effects in the Optimization of 3-Methyl-1,2,3,4-tetrahydroisoquinoline Inhibitors by β-FluorinationGrunewald, Gary L.; Seim, Mitchell R.; Lu, Jian; Makboul, Mariam; Criscione, Kevin R.Journal of Medicinal Chemistry (2006), 49 (10), 2939-2952CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)3-Methyl-1,2,3,4-tetrahydroisoquinolines (3-methyl-THIQs) are potent inhibitors of phenylethanolamine N-methyltransferase (PNMT), but are not selective due to significant affinity for the α2-adrenoceptor. Fluorination of the Me group lowers the pKa of the THIQ amine from 9.53 (CH3) to 7.88 (CH2F), 6.42 (CHF2), and 4.88 (CF3). This decrease in pKa results in a redn. in affinity for the α2-adrenoceptor. However, increased fluorination also results in a redn. in PNMT inhibitory potency, apparently due to steric and electrostatic factors. Biochem. evaluation of a series of 3-fluoromethyl-THIQs and 3-trifluoromethyl-THIQs showed that the former were highly potent inhibitors of PNMT, but were often nonselective due to significant affinity for the α2-adrenoceptor, while the latter were devoid of α2-adrenoceptor affinity, but also lost potency at PNMT. 3-Difluoromethyl-7-substituted-THIQs have the proper balance of both steric and pKa properties and thus have enhanced selectivity vs. the corresponding 3-fluoromethyl-7-substituted-THIQs and enhanced PNMT inhibitory potency vs. the corresponding 3-trifluoromethyl-7-substituted-THIQs. Using the "Goldilocks Effect" analogy, the 3-fluoromethyl-THIQs are too potent (too hot) at the α2-adrenoceptor and the 3-trifluoromethyl-THIQs are not potent enough (too cold) at PNMT, but the 3-difluoromethyl-THIQs are just right. They are both potent inhibitors of PNMT and highly selective due to low affinity for the α2-adrenoceptor. This seems to be the first successful use of the β-fluorination of aliph. amines to impart selectivity to a pharmacol. agent while maintaining potency at the site of interest.268Xu, S.; Zhu, B.; Teffera, Y.; Pan, D. E.; Caldwell, C. G.; Doss, G.; Stearns, R. A.; Evans, D. C.; Beconi, M. G. Metabolic activation of fluoropyrrolidine dipeptidyl peptidase-IV inhibitors by rat liver microsomes. Drug Metab. Dispos. 2005, 33, 121– 130, DOI: 10.1124/dmd.104.001842[Crossref], [PubMed], [CAS], Google Scholar268https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXltlensg%253D%253D&md5=2422da6be675647a96f6b76ab1e88a84Metabolic activation of fluoropyrrolidine dipeptidyl peptidase-IV inhibitors by rat liver microsomesXu, Shiyao; Zhu, Bing; Teffera, Yohannes; Pan, Deborah E.; Caldwell, Charles G.; Doss, George; Stearns, Ralph A.; Evans, David C.; Beconi, Maria G.Drug Metabolism and Disposition (2005), 33 (1), 121-130CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)The current study evaluated the potential for two dipeptidyl peptidase-IV (DPP-IV) inhibitor analogs (1S)-1-(trans-4-{[(4-trifluoromethoxyphenyl)sulfonyl]amino}cyclohexyl)-2-[(3S)-3-fluoropyrrolidin-1-yl]-2-oxoethanaminium chloride and (1S)-1-(trans-4-{[(2,4-difluorophenyl)sulfonyl]amino}cyclohexyl)-2-[(3S)-3-fluoropyrrolidin-1-yl]-2-oxoethanaminium chloride (MRL-A and MRL-B), contg. a fluoropyrrolidine moiety in the structure, to undergo metabolic activation. The irreversible binding of these tritium-labeled compds. to rat liver microsomal protein was time- and NADPH-dependent and was attenuated by the addn. of reduced glutathione (GSH) or N-acetylcysteine (NAC) to the incubation, indicating that chem. reactive intermediates were formed and trapped by these nucleophiles. Mass spectrometric analyses and further trapping expts. with semicarbazide indicated that the fluoropyrrolidine ring had undergone sequential oxidn. and defluorination events resulting in the formation of GSH or NAC conjugates of the pyrrolidine moiety. The bioactivation of MRL-A was catalyzed primarily by rat recombinant CYP3A1 and CYP3A2. Pretreatment of rats with prototypic CYP3A1 and 3A2 inducers (pregnenolone-16α-carbonitrile and dexamethasone) enhanced the extent of bioactivation which, in turn, led to a higher degree of in vitro irreversible binding to microsomal proteins (5- and 9-fold increase, resp.). Herein, the authors describe studies that demonstrate that the fluoropyrrolidine ring is prone to metabolic activation and that GSH or NAC can trap the reactive intermediates to form adducts that provide insight into the mechanisms of bioactivation.269Edmondson, S. D.; Mastracchio, A.; Mathvink, R. J.; He, J.; Harper, B.; Park, Y. J.; Beconi, M.; Di Salvo, J.; Eiermann, G. J.; He, H.; Leiting, B.; Leone, J. F.; Levorse, D. A.; Lyons, K.; Patel, R. A.; Patel, S. B.; Petrov, A.; Scapin, G.; Shang, J.; Roy, R. S.; Smith, A.; Wu, J. K.; Xu, S.; Zhu, B.; Thornberry, N. A.; Weber, A. E. (2S,3S)-3-Amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N-dimethyl-4-oxo-2-(4-[1,2,4]triazolo[1,5-a]-pyridin-6-ylphenyl)butanamide: a selective α-amino amide dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. J. Med. Chem. 2006, 49, 3614– 3627, DOI: 10.1021/jm060015t[ACS Full Text], [CAS], Google Scholar269https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XkslKht7Y%253D&md5=6ebe737acc317676ca1c7e4178d9719f(2S,3S)-3-Amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N-dimethyl-4-oxo-2-(4-[1,2,4]triazolo[1,5-a]- pyridin-6-ylphenyl)butanamide: A selective α-amino amide dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetesEdmondson, Scott D.; Mastracchio, Anthony; Mathvink, Robert J.; He, Jiafang; Harper, Bart; Park, You-Jung; Beconi, Maria; Di Salvo, Jerry; Eiermann, George J.; He, Huaibing; Leiting, Barbara; Leone, Joseph F.; Levorse, Dorothy A.; Lyons, Kathryn; Patel, Reshma A.; Patel, Sangita B.; Petrov, Aleksandr; Scapin, Giovanna; Shang, Jackie; Roy, Ranabir Sinha; Smith, Aaron; Wu, Joseph K.; Xu, Shiyao; Zhu, Bing; Thornberry, Nancy A.; Weber, Ann E.Journal of Medicinal Chemistry (2006), 49 (12), 3614-3627CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of β-substituted biarylphenylalanine amides were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-4) for the treatment of type 2 diabetes. Optimization of the metabolic profile of early analogs led to the discovery of (2S,3S)-3-amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N-dimethyl-4-oxo-2-(4-[1,2,4]triazolo[1,5-a]pyridin-6-ylphenyl)butanamide I, a potent, orally active DPP-4 inhibitor (IC50 = 6.3 nM) with excellent selectivity, oral bioavailability in preclin. species, and in vivo efficacy in animal models. Compd. I was selected for further characterization as a potential new treatment for type 2 diabetes.270Sharma, R.; Sun, H.; Piotrowski, D. W.; Ryder, T. F.; Doran, S. D.; Dai, H.; Prakash, C. Metabolism, excretion, and pharmacokinetics of ((3,3-difluoropyrrolidin-1-yl)((2S,4S)-4-(4-(pyrimidin-2-yl)piperazin-1-yl)pyrrolidin-2-yl)methanone, a dipeptidyl peptidase inhibitor, in rat, dog and human. Drug Metab. Dispos. 2012, 40, 2143– 2161, DOI: 10.1124/dmd.112.047316[Crossref], [PubMed], [CAS], Google Scholar270https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFKkt7rP&md5=8fa2c39deb0ffc3dcda00ee7aa68e250Metabolism, excretion, and pharmacokinetics of ((3,3-difluoropyrrolidin-1-yl)((2S,4S)-4-(4-(pyrimidin-2-yl)piperazin-1-yl)pyrrolidin-2-yl)methanone, a dipeptidyl peptidase inhibitor, in rat, dog and humanSharma, Raman; Sun, Hao; Piotrowski, David W.; Ryder, Tim F.; Doran, Shawn D.; Dai, Haiqing; Prakash, ChandraDrug Metabolism & Disposition (2012), 40 (11), 2143-2161CODEN: DMDSAI; ISSN:1521-009X. (American Society for Pharmacology and Experimental Therapeutics)The disposition of 3,3-difluoropyrrolidin-1-yl{(2S,4S)-4-[4-(pyrimidin-2-yl)piperazin-1-yl]pyrrolidin-2-yl}methanone (PF-00734200), a dipeptidyl peptidase IV inhibitor that progressed to phase 3 for the treatment of type 2 diabetes, was examd. in rats, dogs, and humans after oral administration of a single dose of [14C]PF-00734200. Mean recoveries of administered radioactivity were 97.1, 92.2, and 87.2% in rats, dogs, and humans, resp. The majority of radioactive dose was detected in the urine of dogs and humans and in the feces of rats. Absorption of PF-00734200 was rapid in all species, with maximal plasma concns. of radioactivity achieved within 1 h after the dose. Circulating radioactivity was primarily composed of the parent drug (79.9, 80.2, and 94.4% in rat, dog, and human, resp.). The major route of metab. was due to hydroxylation at the 5' position of the pyrimidine ring (M5) in all species. In vitro expts. with recombinant cytochrome P 450 isoforms suggested that the formation of M5 was catalyzed both by CYP2D6 and CYP3A4. Mol. docking simulations showed that the 5' position of the pyrimidine moiety of PF-00734200 can access the heme iron-oxo of both CYP3A4 and CYP2D6 in an energetically favored orientation. Other metabolic pathways included amide hydrolysis (M2), N-dealkylation at the piperazine nitrogen (M3) and an unusual metabolite resulting from scission of the pyrimidine ring (M1). Phase II metabolic pathways included the following: carbamoyl glucuronidation (M9), glucosidation (M15) on the pyrrolidine nitrogen, and conjugation with creatinine to form an unusual metabolite/metabonate (M16). The data from these studies suggest that PF-00734200 is eliminated by both metab. and renal clearance.271Tremblay, M.; Bethell, R. C.; Cordingley, M. G.; DeRoy, P.; Duan, J.; Duplessis, M.; Edwards, P. J.; Faucher, A. M.; Halmos, T.; James, C. A.; Kuhn, C.; Lacoste, J. E.; Lamorte, L.; LaPlante, S. R.; Malenfant, E.; Minville, J.; Morency, L.; Morin, S.; Rajotte, D.; Salois, P.; Simoneau, B.; Tremblay, S.; Sturino, C. F. Identification of benzofurano[3,2-d]pyrimidin-2-ones, a new series of HIV-1 nucleotide-competing reverse transcriptase inhibitors. Bioorg. Med. Chem. Lett. 2013, 23, 2775– 2780, DOI: 10.1016/j.bmcl.2013.02.042[Crossref], [PubMed], [CAS], Google Scholar271https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXktlCmurk%253D&md5=7dc770482e4deef5332c06b6a31e8a8eIdentification of benzofurano[3,2-d]pyrimidin-2-ones, a new series of HIV-1 nucleotide-competing reverse transcriptase inhibitorsTremblay, Martin; Bethell, Richard C.; Cordingley, Michael G.; DeRoy, Patrick; Duan, Jianmin; Duplessis, Martin; Edwards, Paul J.; Faucher, Anne-Marie; Halmos, Ted; James, Clint A.; Kuhn, Cyrille; Lacoste, Jean-Eric; Lamorte, Louie; LaPlante, Steven R.; Malenfant, Eric; Minville, Joannie; Morency, Louis; Morin, Sebastien; Rajotte, Daniel; Salois, Patrick; Simoneau, Bruno; Tremblay, Sonia; Sturino, Claudio F.Bioorganic & Medicinal Chemistry Letters (2013), 23 (9), 2775-2780CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Screening of our sample collection led to the identification of a set of benzofurano[3,2-d]pyrimidine-2-one hits acting as nucleotide-competing HIV-1 reverse transcriptase inhibitors (NcRTI). Significant improvement in antiviral potency was achieved when substituents were introduced at positions N1, C4, C7 and C8 on the benzofuranopyrimidone scaffold. The series was optimized from low micromolar enzymic activity against HIV-1 RT and no antiviral activity to low nanomolar antiviral potency. Further profiling of inhibitor 30 showed promising overall in vitro properties and also demonstrated that its potency was maintained against viruses resistant to the other major classes of HIV-1 RT inhibitors.272Wang, X.; Sun, M.; New, C.; Nam, S.; Blackaby, W. P.; Hodges, A. J.; Nash, D.; Matteucci, M.; Lyssikatos, J. P.; Fan, P. W.; Tay, S.; Chang, J. H. Probing mechanisms of CYP3A time-dependent inhibition using a truncated model system. ACS Med. Chem. Lett. 2015, 6, 925– 929, DOI: 10.1021/acsmedchemlett.5b00191[ACS Full Text], [CAS], Google Scholar272https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFGitb%252FI&md5=02eb92abebe87a4e406ca9a6241a0471Probing Mechanisms of CYP3A Time-Dependent Inhibition Using a Truncated Model SystemWang, Xiaojing; Sun, Minghua; New, Connie; Nam, Spencer; Blackaby, Wesley P.; Hodges, Alastair J.; Nash, David; Matteucci, Mizio; Lyssikatos, Joseph P.; Fan, Peter W.; Tay, Suzanne; Chang, Jae H.ACS Medicinal Chemistry Letters (2015), 6 (8), 925-929CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)Time-dependent inhibition (TDI) of cytochrome P 450 (CYP) enzymes may incur serious undesirable drug-drug interactions and in rare cases drug-induced idiosyncratic toxicity. The reactive metabolites are often generated through multiple sequential biotransformations and form adducts with CYP enzymes to inactivate their function. The complexity of these processes makes addressing TDI liability very challenging. Strategies to mitigate TDI are therefore highly valuable in discovering safe therapies to benefit patients. In this Letter, the authors disclose the simplified approach toward addressing CYP3A TDI liabilities, guided by metabolic mechanism hypotheses. By adding a Me group onto the α carbon of a basic amine, TDI activities of both the truncated and full mols. I and II were completely eliminated. The authors propose that truncated mols., albeit with caveats, may be used as surrogates for full mols. to investigate TDI.273Wang, X.; Blackaby, W.; Allen, V.; Chan, G. K. Y.; Chang, J. H.; Chiang, P. C.; Diene, C.; Drummond, J.; Do, S.; Fan, E.; Harstad, E. B.; Hodges, A.; Hu, H.; Jia, W.; Kofie, W.; Kolesnikov, A.; Lyssikatos, J. P.; Ly, J.; Matteucci, M.; Moffat, J. G.; Munugalavadla, V.; Murray, J.; Nash, D.; Noland, C. L.; Del Rosario, G.; Ross, L.; Rouse, C.; Sharpe, A.; Slaga, D.; Sun, M.; Tsui, V.; Wallweber, H.; Yu, S. F.; Ebens, A. J. Optimization of pan-Pim kinase activity and oral bioavailability leading to diaminopyrazole (GDC-0339) for the treatment of multiple myeloma. J. Med. Chem. 2019, 62, 2140– 2153, DOI: 10.1021/acs.jmedchem.8b01857[ACS Full Text], [CAS], Google Scholar273https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXisVSqt78%253D&md5=bf8f5e5ac1f4cc29d1b62dcd72e7a815Optimization of Pan-Pim Kinase Activity and Oral Bioavailability Leading to Diaminopyrazole (GDC-0339) for the Treatment of Multiple MyelomaWang, Xiaojing; Blackaby, Wesley; Allen, Vivienne; Chan, Grace Ka Yan; Chang, Jae H.; Chiang, Po-Chang; Diene, Coura; Drummond, Jason; Do, Steven; Fan, Eric; Harstad, Eric B.; Hodges, Alastair; Hu, Huiyong; Jia, Wei; Kofie, William; Kolesnikov, Aleksandr; Lyssikatos, Joseph P.; Ly, Justin; Matteucci, Mizio; Moffat, John G.; Munugalavadla, Veerendra; Murray, Jeremy; Nash, David; Noland, Cameron L.; Del Rosario, Geoff; Ross, Leanne; Rouse, Craig; Sharpe, Andrew; Slaga, Dionysos; Sun, Minghua; Tsui, Vickie; Wallweber, Heidi; Yu, Shang-Fan; Ebens, Allen J.Journal of Medicinal Chemistry (2019), 62 (4), 2140-2153CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Pim kinases have been targets of interest for a no. of therapeutic areas. Evidence of durable single-agent efficacy in human clin. trials validated Pim kinase inhibition as a promising therapeutic approach for multiple myeloma patients. Here, we report the compd. optimization leading to GDC-0339 (16), a potent, orally bioavailable, and well tolerated pan-Pim kinase inhibitor that proved efficacious in RPMI8226 and MM.1S human multiple myeloma xenograft mouse models and has been evaluated as an early development candidate.274Acton, B.; Small, H. F.; Smith, K. M.; McGonagle, A.; Stowell, A. I. J.; James, D. I.; Hamilton, N. M.; Hamilton, N.; Hitchin, J. R.; Hutton, C. P.; Waddell, I. D.; Ogilvie, D. J.; Jordan, A. M. Fluoromethylcyclopropylamine derivatives as potential in vivo toxicophores - A cautionary disclosure. Bioorg. Med. Chem. Lett. 2019, 29, 560– 562, DOI: 10.1016/j.bmcl.2018.12.066[Crossref], [PubMed], [CAS], Google Scholar274https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXkvVegtw%253D%253D&md5=041dbec76f5fbff64ea01e59315cfa2fFluoromethylcyclopropylamine derivatives as potential in vivo toxicophores - A cautionary disclosureActon, Ben; Small, Helen F.; Smith, Kate M.; McGonagle, Alison; Stowell, Alexandra I. J.; James, Dominic I.; Hamilton, Niall M.; Hamilton, Nicola; Hitchin, James R.; Hutton, Colin P.; Waddell, Ian D.; Ogilvie, Donald J.; Jordan, Allan M.Bioorganic & Medicinal Chemistry Letters (2019), 29 (4), 560-562CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Fluorination of metabolic hotspots in a mol. is a common medicinal chem. strategy to improve in vivo half-life and exposure and, generally, this strategy offers significant benefits. Here, we report the application of this strategy to a series of poly-ADP ribose glycohydrolase (PARG) inhibitors, resulting in unexpected in vivo toxicity which was attributed to this single-atom modification.275Berkowitz, D. B.; Karukurichi, K. R.; de la Salud-Bea, R.; Nelson, D. L.; McCune, C. D. Use of fluorinated functionality in enzyme inhibitor development: mechanistic and analytical advantages. J. Fluorine Chem. 2008, 129, 731– 742, DOI: 10.1016/j.jfluchem.2008.05.016[Crossref], [PubMed], [CAS], Google Scholar275https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtVyitrnK&md5=87124626a1eceb76440c42fd87cc3360Use of fluorinated functionality in enzyme inhibitor development: Mechanistic and analytical advantagesBerkowitz, David B.; Karukurichi, Kannan R.; de la Salud-Bea, Roberto; Nelson, David L.; McCune, Christopher D.Journal of Fluorine Chemistry (2008), 129 (9), 731-742CODEN: JFLCAR; ISSN:0022-1139. (Elsevier B.V.)A review. On the one hand, owing to its electronegativity, relatively small size, and notable leaving group ability from anionic intermediates, fluorine offers unique opportunities for mechanism-based enzyme inhibitor design. On the other, the "bio-orthogonal" and NMR-active 19-fluorine nucleus allows the bioorg. chemist to follow the mechanistic fate of fluorinated substrate analogs or inhibitors as they are enzymically processed. This article takes an overview of the field, highlighting key developments along these lines. It begins by highlighting new screening methodologies for drug discovery that involve appropriate tagging of either the substrate or an array of potential substrates (i.e., in proteomics screens) with 19F-markers that then report back on turnover and function, resp., via the NMR screen. Taking this one step further, substrate-tagging with fluorine can be done in such a manner as to provide stereochem. information on enzyme mechanism. For example, substitution of one of the terminal hydrogens in phosphoenolpyruvate, provides insight into the, otherwise latent, facial selectivity of C-C bond formation in KDO synthase. Perhaps, most importantly, from the point of view of this discussion, appropriately tailored fluorinated functionality can be used to form stabilized "transition state analog" complexes with target enzymes. Thus, 5-fluorinated pyrimidines, α-fluorinated ketones, and 2-fluoro-2-deoxysugars each lead to covalent adduction of catalytic active site residues in thymidylate synthase (TS), serine protease and glycosidase enzymes, resp. In all such cases, 19F NMR allows the bioorg. chemist to spectrally follow "transition state analog" formation. Finally, the use of specific fluorinated functionality to engineer "suicide substrates" is highlighted in a discussion of the development of the α-(2'Z-fluoro)vinyl trigger for amino acid decarboxylase inactivation. Here 19F NMR allows the bioorg. chemist to glean useful partition ratio data directly from the NMR tube.276Conti, P.; Tamborini, L.; Pinto, A.; Blondel, A.; Minoprio, P.; Mozzarelli, A.; De Micheli, C. Drug discovery targeting amino acid racemases. Chem. Rev. 2011, 111, 6919– 6946, DOI: 10.1021/cr2000702[ACS Full Text], [CAS], Google Scholar276https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFGqurjL&md5=33df0c7a9ed05e54141c7b3f13203954Drug Discovery Targeting Amino Acid RacemasesConti, Paola; Tamborini, Lucia; Pinto, Andrea; Blondel, Arnaud; Minoprio, Paola; Mozzarelli, Andrea; De Micheli, CarloChemical Reviews (Washington, DC, United States) (2011), 111 (11), 6919-6946CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. In the following chapters we will discuss the classification of amino acid racemases, involved in the prodn. of D-amino acids through a racemization process of the L counterparts, their diffusion, function, localization, structural features and the state of the art in the discovery of novel drugs targeting such enzymes for the treatment of different pathologies.277Azam, M. A.; Jayaram, U. Inhibitors of alanine racemase enzyme: a review. J. Enzyme Inhib. Med. Chem. 2016, 31, 517– 526, DOI: 10.3109/14756366.2015.1050010[Crossref], [PubMed], [CAS], Google Scholar277https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmvFCmurg%253D&md5=26a4555f21c235bab0797409aec6902eInhibitors of alanine racemase enzyme: a reviewAzam, Mohammed Afzal; Jayaram, UnniJournal of Enzyme Inhibition and Medicinal Chemistry (2016), 31 (4), 517-526CODEN: JEIMAZ; ISSN:1475-6366. (Taylor & Francis Ltd.)Alanine racemase is a fold type III PLP-dependent amino acid racemase enzyme catalyzing the conversion of L-alanine to D-alanine utilized by bacterial cell wall for peptidoglycan synthesis. As there are no known homologs in humans, it is considered as an excellent antibacterial drug target. The std. inhibitors of this enzyme include O-carbamyl-D-serine, D-cycloserine, chlorovinyl glycine, alaphosphin, etc. D-Cycloserine is indicated for pulmonary and extra pulmonary tuberculosis but therapeutic use of drug is limited due to its severe toxic effects. Toxic effects due to off-target affinities of cycloserine and other substrate analogs have prompted new research efforts to identify alanine racemase inhibitors that are not substrate analogs. In this review, an updated status of known inhibitors of alanine racemase enzyme has been provided which will serve as a rich source of structural information and will be helpful in generating selective and potent inhibitor of alanine racemase.278Kollonitsch, J.; Barash, L.; Kahan, F. M.; Kropp, H. New antibacterial agent via photofluorination of a bacterial cell wall constituent. Nature 1973, 243, 346– 347, DOI: 10.1038/243346a0[Crossref], [PubMed], [CAS], Google Scholar278https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3sXkslCitb8%253D&md5=64452355855203c632f72c38e70ba7c8New antibacterial agent via photofluorination of a bacterial cell wall constituentKollonitsch, J.; Barash, L.; Kahan, F. M.; Kropp, H.Nature (London, United Kingdom) (1973), 243 (5406), 346-7CODEN: NATUAS; ISSN:0028-0836.A highly active antibacterial agent, which is active against gram-neg. and pos. bacteria, was prepd. by C-fluorination of a key component of the bacterial cell wall, D-alanine [338-69-2]. Thus, 3-fluoro-D-alanine [35455-20-0] at concns. of 6-100 μg/ml inhibited the growth of Escherichia coli. D-alanine at 6-100 μg/ml reversed the inhibitory effect of 3-fluoro-D-alanine at 25 μg/ml. The ED50 values of 3-fluoro-D-alanine for Streptococcus pyrogenes and Diplococcus pneumoniae in mice were 1.1 and 5 mg/kg, resp. Mice survived a single, oral dose of 2 g/kg.279Kollonitsch, J.; Barash, L. Organofluorine synthesis via photofluorination: 3-fluoro-d-alanine and 2-deuterio analogue, antibacterials related to the bacterial cell wall. J. Am. Chem. Soc. 1976, 98, 5591– 5593, DOI: 10.1021/ja00434a030[ACS Full Text], [CAS], Google Scholar279https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE28XlsVentro%253D&md5=15d16fed9c9c23e8206dbff5abf0cc71Organofluorine synthesis via photofluorination: 3-fluoro-D-alanine and 2-deuterio analog, antibacterials related to the bacterial cell wallKollonitsch, J.; Barash, L.Journal of the American Chemical Society (1976), 98 (18), 5591-3CODEN: JACSAT; ISSN:0002-7863.The antibacterials 3-fluoro-D-alanine (I) and its 2-deuterated version (II) were prepd. The design of I exploits a fundamental divergence in biosynthesis of the peptidoglycan component of the bacterial cell wall and of the metabolic pathways in humans. This divergence suggested application of the concept of antimetabolite synthesis via the specific approach of photofluorination. Thus, photofluorination of(D-alanine generated I which displays a high degree of antibacterial activity. A variant of I increased metabolic stability-and with unimpaired antibacterial activity-was obtained via photofluorination of 2-deuterio-D-alanine, namely 3-fluoro-D-alanine-2d (II), effective in vitro and in vivo against every bacterial strain tested.280Silverman, R. B.; Abeles, R. H. Inactivation of pyridoxal phosphate dependent enzymes by mono- and polyhaloalanines. Biochemistry 1976, 15, 4718– 4723, DOI: 10.1021/bi00666a028[ACS Full Text], [CAS], Google Scholar280https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2sXhsFyj&md5=4d136781d42b7741be7a8ebbf04de1e2Inactivation of pyridoxal phosphate dependent enzymes by mono- and polyhaloalaninesSilverman, Richard B.; Abeles, Robert H.Biochemistry (1976), 15 (21), 4718-23CODEN: BICHAW; ISSN:0006-2960.β,β-Dichloro- and β,β,β-trifluoroalanine irreversibly inactivate a no. of pyridoxal phosphate-dependent enzymes which catalyze β- or γ-elimination reactions. The inactivation is time dependent and the rate of inactivation is 1st order in enzyme concn. This suggests that inactivation is due to covalent modification of the enzyme by a species generated at the active site from the polyhaloalanine (i.e., suicide inactivation). Monohaloalanines are substrates and do not inactivate. For γ-cystathionase, covalent and stoichiometric attachment of β,β,β-trifluoroalanine-1-14C was shown. It is proposed that the mechanism of inactivation involves Schiff base formation between inactivator and enzyme-bound pyridoxal and subsequent elimination of HCl from dichloroalanine or HF from trifluoroalanine. This results in the formation of a β-halo-α,β unsatd. imine, an activated Michael acceptor. Michael addition of a nucleophile at the active site leads to covalent labeling of the enzyme and inactivation. Alanine racemase is also inactivated by the 2 polyhaloalanines. Glutamate-pyruvate and glutamate-oxalocetate transaminases are inactivated by monohaloalanines but not by polyhaloalanines.281Wang, E.; Walsh, C. Suicide substrates for the alanine racemase of Escherichia coli B. Biochemistry 1978, 17, 1313– 1321, DOI: 10.1021/bi00600a028[ACS Full Text], [CAS], Google Scholar281https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXktVahs7c%253D&md5=0d32c8dfec57779e35308e0c360e3f06Suicide substrates for the alanine racemase of Escherichia coli BWang, Elizabeth; Walsh, ChristopherBiochemistry (1978), 17 (7), 1313-21CODEN: BICHAW; ISSN:0006-2960.Alanine racemase was purified ∼9000-fold from E. coli and found to be a dimer of 100,000 daltons, contg. 1 mol. pyridoxal phosphate/subunit. The mol. basis for irrversible active site-directed inactivation of the enzyme by a no. of suicide substrates was examd. Both D and L isomers of β-fluoroalanine and β-chloroalanine partition between (a) α,β elimination to pyruvate, ammonia, and halide ion or (b) inactivation. No racemization is detectable. The Vmax for pyruvate formation from L-chloroalanine is ∼50-fold lower than from the D isomer of chloroalanine or either fluoroalanine. However, both enantiomeric pairs partition identically, ∼830 turnovers/inactivating event. This invariant partition ratio suggests that a common intermediate, the eneamino acid-pyridoxal phosphate complex, is the species responsible for inactivation, probably by Michael attack from a nucleophilic residue at the enzyme active site. In keeping with this idea, O-carbamoyl-D-serine and O-acetyl-D-serine also undergo enzyme-catalyzed elimination for 830 turnovers before causing irreversible inactivation, presumably from the same intermediate. In contrast, the L isomers of O-carbamoyl- or O-acetlyserine do not eliminate nor do they induce inactivation, but serve merely as reversible, competitive inhibitors of the enzyme. This suggests asym. binding regions for bulky β substituents at the active site and suggests D isomers of substituted β-alanines would be preferentially effective enzyme inactivators. D-Cycloserine also inactivates alanine racemase in time-dependent fashion. Thus, both natural antibiotics, O-carbamoyl-D-serine and D-cycloserine, previously reported as reversible alanine racemase inhibitors, are in fact suicide substrates along with the β-haloalanines.282Badet, B.; Roise, D.; Walsh, C. T. Inactivation of the dadB Salmonella typhimurium alanine racemase by D and L isomers of β-substituted alanines: kinetics, stoichiometry, active site peptide sequencing, and reaction mechanism. Biochemistry 1984, 23, 5188– 5194, DOI: 10.1021/bi00317a016[ACS Full Text], [CAS], Google Scholar282https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXlvVKru74%253D&md5=d55fa0aee74737d76947707917b98c66Inactivation of the dadB Salmonella typhimurium alanine racemase by D and L isomers of β-substituted alanines: kinetics, stoichiometry, active site peptide sequencing, and reaction mechanismBadet, Bernard; Roise, David; Walsh, Christopher T.Biochemistry (1984), 23 (22), 5188-94CODEN: BICHAW; ISSN:0006-2960.The pyridoxal phosphate-dependent S. typhimurium gene dadb alanine racemase (I) was inactivated with D- and L-β-fluoroalanine, D- and L-β-chloroalanine, and O-acetyl-D-serine. I inactivation with each isomer of β-chloro[14C]alanine followed by NaBH4 redn. and trypsin digestion afforded a single radiolabeled peptide. In the same manner, NaB3H4-reduced native I gave a single labeled peptide after trypsin digestion. Purifn. and sequencing of these 3 radioactive peptides revealed them to be a common, unique hexadecapeptide which contained labeled lysine at position 6 in each case. I which had been inactivated, but not reductively stabilized with NaBH4, released a labile pyridoxal phosphate-inactivator adduct on denaturation. The structure of this adduct suggested that I was inactivated by trapping the coenzyme in a ternary adduct with inactivator and the active-site lysine. Under denaturing conditions, facile α,β-elimination occurred, releasing the aldol adduct of pyruvate and pyridoxal phosphate. Redn. of the ternary enzyme adduct blocked this elimination pathway. The overall mechanism of I inactivation was discussed in light of these results.283Wang, E. A.; Walsh, C. Characteristics of β,β-difluoroalanine and β,β,β-trifluoroalanine as suicide substrates for Escherichia coli B alanine racemase. Biochemistry 1981, 20, 7539– 7546, DOI: 10.1021/bi00529a032[ACS Full Text], [CAS], Google Scholar283https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XivVSg&md5=d4db063b01c8f5861db1032c0ede2c5dCharacteristics of β,β-difluoroalanine and β,β,β-trifluoroalanine as suicide substrates for E. coli B alanine racemaseWang, Elizabeth A.; Walsh, ChristopherBiochemistry (1981), 20 (26), 7539-46CODEN: BICHAW; ISSN:0006-2960.Alanine racemase from Escherichia coli B has been shown to process D- and L-isomers of β-fluoroalanine as suicide substrates with an identical partitioning ratio for each enantiomer of 820 catalytic eliminations of HF per enzymic inactivation event, suggesting the aminoacrylate-pyridoxal phosphate (PLP) complex as a common, sym. partitioning species. In an attempt to systematically vary the partition ratio, an index of killing efficiency, the β,β-difluoroalanine and β,β,β-trifluoroalanine isomers were evaluated for substrate processing, suicidal inactivation kinetics, and partitioning ratio, and stability of inactive , derivatized enzyme forms. Both difluoroalanine isomers showed high Km values (116 mM for D, 102 mM for L) in catalytic HF loss to form fluoropyruvate. The Vmax for the D-isomer was ∼14-fold higher than that of the L-isomer. Limiting inactivation rate consts., calcd. from kcat and obsd. partition ratios of 5000 and 2600, resp., were 2.2 min-1 for D-difluoroalanine and 0.33 min-1 for L-difluoroalanine. For comparison, DL-trifluoroalanine turned over <10 times/enzyme mol. inactivated and was thus a very efficient suicide substrate. The est. inactivation rate const. was <1.0 min-1. These data were analyzed in terms of the partitioning behavior of the monofluoro- and difluoroaminoacrylate-PLP complexes as partitioning intermediates for turnover or for racemase inactivation. Whereas the mono- and trifluoroalanines yield stable inactive species the difluoroalanine isomers produced labile enzyme derivs., and regain of catalytic activity was analyzed in terms of the anticipated oxidn. state at the β-C atom of the substrate fragment adducted to the enzyme.284Faraci, W. S.; Walsh, C. T. Mechanism of inactivation of alanine racemase by β,β,β-trifluoroalanine. Biochemistry 1989, 28, 431– 437, DOI: 10.1021/bi00428a004[ACS Full Text], [CAS], Google Scholar284https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXltlGgtQ%253D%253D&md5=36b00328b07b93dbb9daff89ee5e8d3fMechanism of inactivation of alanine racemase by β,β,β-trifluoroalanineFaraci, W. Stephen; Walsh, Christopher T.Biochemistry (1989), 28 (2), 431-7CODEN: BICHAW; ISSN:0006-2960.The alanine racemases are a group of pyridoxal phosphate (PLP)-dependent bacterial enzymes that catalyze the racemization of alanine, providing D-alanine for cell wall synthesis. The inactivation of the alanine racemases from the Gram-neg. organism, Salmonella typhimurium, and the Gram-pos. organism, Bacillus stearothermophilus, with β,β,β-trifluoroalanine was studied. The inactivation occurred with the same rate const. as that for formation of a broad 460-490-nm chromophore. The loss of 2 F-/mol of inactivated enzyme and the retention of the [1-14C]trifluoroalanine label accompanied inhibition, suggesting a monofluoro enzyme adduct. Partial denaturation (1M guanidine) led to rapid return of the initial 420-nm chromophore, followed by a slower (t1/2 ∼30 min-1 h) loss of F- and 14CO2 release. At this point, redn. by NaB3H4 and tryptic digestion yielded a single radiolabeled peptide. The purifn. and sequencing of the peptide revealed that lysine-38 was covalently attached to the PLP cofactor. A mechanism for enzyme inactivation by trifluoroalanine is proposed and contrasted with earlier results on monohaloalanines, in which nucleophilic attack of released aminoacrylate on the PLP aldimine leads to enzyme inactivation. For trifluoroalanine inactivation, nucleophilic attack of lysine-38 on the electrophilic β-difluoro-α,β-unsatd. imine provides an alternative mode of inhibition for these enzymes.285Thornberry, N. A.; Bull, H. G.; Taub, D.; Wilson, K. E.; Giménez-Gallego, G.; Rosegay, A.; Soderman, D. D.; Patchett, A. A. Mechanism-based inactivation of alanine racemase by 3-halovinylglycines. J. Biol. Chem. 1991, 266, 21657– 21665[PubMed], [CAS], Google Scholar285https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXmslChu7Y%253D&md5=c9a822e5b5c446018a29c0d961411daaMechanism-based inactivation of alanine racemase by 3-halovinylglycinesThornberry, Nancy A.; Bull, Herbert G.; Taub, David; Wilson, Kenneth E.; Gimenez-Gallego, Guillermo; Rosegay, Avery; Soderman, Denis D.; Patchett, Arthur A.Journal of Biological Chemistry (1991), 266 (32), 21657-65CODEN: JBCHA3; ISSN:0021-9258.Alanine racemase, an enzyme important to bacterial cell wall synthesis, is irreversibly inactivated by 3-chloro- and 3-fluorovinylglycine. Using alanine racemase purified to homogeneity from Escherichia coli B, the efficient inactivation produced a lethal event for every 2.2 nonlethal turnovers, compared to 1 in 800 for fluoroalanine. The mechanism of inhibition involves enzyme-catalyzed halide elimination to form an allenic intermediate that partitions between reversible and irreversible covalent adducts, in the ratio 3:7. The reversible adduct (λmax = 516 nm) decays to regenerate free enzyme with a half-life of 23 min. The lethal event involves irreversible alkylation of a tyrosine residue in the sequence -Val-Gly-Tyr-Gly-Gly-Arg-: the second-order rate const. for this process with D-chlorovinylglycine (122 M-1 s-1), the most reactive analog examd., is faster than the equiv. rate const. for D-fluoroalanine (93 M-1 s-1). The high killing efficiency and fast turnover of these mechanism-based inhibitors suggest that their design, employing the haloethylene moiety to generate a reactive allene during catalysis, could be extended to provide useful inhibitors of a variety of enzymes that conduct carbanion chem.286Thornberry, N. A.; Bull, H. G.; Taub, D.; Greenlee, W. J.; Patchett, A. A.; Cordes, E. H. 3-Halovinylglycines. Efficient irreversible inhibitors of E. coli alanine racemase. J. Am. Chem. Soc. 1987, 109, 7543– 7544, DOI: 10.1021/ja00258a056[ACS Full Text], [CAS], Google Scholar286https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXisFGj&md5=e19af51c169636042c9fe6af3ee22d313-Halovinylglycines. Efficient irreversible inhibitors of Escherichia coli alanine racemaseThornberry, N. A.; Bull, H. G.; Taub, D.; Greenlee, W. J.; Patchett, A. A.; Cordes, E. H.Journal of the American Chemical Society (1987), 109 (24), 7543-4CODEN: JACSAT; ISSN:0002-7863.3-Halovinylglycines are a new class of potent mechanism-based irreversible inactivators of alanine racemase. Results with E. coli β-alanine racemase (I) indicate that nearly every catalytic event results in irreversible inactivation of the enzyme. In contrast, mechanism-based inactivation by previously studied β-substituted alanines is much less efficient, requiring ∼800 turnovers with this enzyme to produce 1 irreversible event. The incubation of homogeneous E. coli I with D-chlorovinylglycine, L-chlorovinylglycine, or DL-fluorovinylglycine resulted in irreversible inactivation of the enzyme as demonstrated by the inability of the enzyme to regain catalytic activity after prolonged dialysis. The inactivation kinetics were characterized by rapid, pseudo-1st-order irreversible inhibition of 70% of the enzyme. The 2nd-order rate const. that describes this initial inactivation for D-chlorovinylglycine (122 M-1 s-1) was comparable to the corresponding rate const. for 3-fluoro-D-alanine (93 M-1 s-1). The irreversible inhibition of the remaining enzyme was described by a 1st-order rate const. that was independent of inhibitor concn., halogen, and stereochem. (1.2 × 10-4 s-1). The fluorovinylglycines were 100-fold less reactive than the chlorovinylglycines. Consistent with the greater efficiency and more complex kinetics, there was phys. evidence that the halovinylglycines follow a different mechanism of inhibition than established for their simpler homologs, the β-substituted alanines.287Jung, M. J. Substrates and inhibitors of aromatic amino acid decarboxylase. Bioorg. Chem. 1986, 14, 429– 443, DOI: 10.1016/0045-2068(86)90007-6[Crossref], [CAS], Google Scholar287https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXmtlelsg%253D%253D&md5=d80ae494b692f22f889e5588bc98781fSubstrates and inhibitors of aromatic amino acid decarboxylaseJung, M. J.Bioorganic Chemistry (1986), 14 (4), 429-43CODEN: BOCMBM; ISSN:0045-2068.A review with 71 refs. on the reaction mechanism and specificity of the title mammalian decarboxylase with substrates, substrate analogs, and inhibitors.288Li, T.; Huo, L.; Pulley, C.; Liu, A. Decarboxylation mechanisms in biological system. Bioorg. Chem. 2012, 43, 2– 14, DOI: 10.1016/j.bioorg.2012.03.001[Crossref], [PubMed], [CAS], Google Scholar288https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtFajurvL&md5=ac3fa2546f673f01736184b01f6feb16Decarboxylation mechanisms in biological systemLi, Tingfeng; Huo, Lu; Pulley, Christopher; Liu, AiminBioorganic Chemistry (2012), 43 (), 2-14CODEN: BOCMBM; ISSN:0045-2068. (Elsevier B.V.)A review, examg. the mechanisms propelling cofactor-independent, org. cofactor-dependent and metal-dependent decarboxylase chem. Decarboxylation, the removal of carbon dioxide from org. acids, is a fundamentally important reaction in biol. Numerous decarboxylase enzymes serve as key components of aerobic and anaerobic carbohydrate metab. and amino acid conversion. In the past decade, knowledge of the mechanisms enabling these crucial decarboxylase reactions has continued to expand and inspire. This review focuses on the org. cofactors biotin, flavin, NAD, pyridoxal 5'-phosphate, pyruvoyl, and thiamin pyrophosphate as catalytic centers. Significant attention is also placed on the metal-dependent decarboxylase mechanisms.289Flynn, G. A.; Beight, D. W.; Bohme, E. H. W.; Metcalf, B. W. The synthesis of fluorinated aminophosphonic acid inhibitors of alanine racemase. Tetrahedron Lett. 1985, 26, 285– 288, DOI: 10.1016/S0040-4039(01)80798-X[Crossref], [CAS], Google Scholar289https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXltVGhtLY%253D&md5=20f34c6b8a9f12295b0e45cf9cde8021The synthesis of fluorinated aminophosphonic acid inhibitors of alanine racemaseFlynn, Gary A.; Beight, Douglas W.; Bohme, Ekkehard H. W.; Metcalf, Brian W.Tetrahedron Letters (1985), 26 (3), 285-8CODEN: TELEAY; ISSN:0040-4039.The synthesis of H2NCHRPO3H2 (I; R = CF3, CHF2, CH2F) from fluorinated acetic acids is described. I are potential inhibitors of alanine racemase which might exhibit the enhanced binding affinity and specificity of H2NCHMeCO2H while retaining the irreversible vector of the fluorinated alanines.290Kollonitsch, J.; Perkins, L. M.; Patchett, A. A.; Doldouras, G. A.; Marburg, S.; Duggan, D. E.; Maycock, A. L.; Aster, S. D. Selective inhibitors of biosynthesis of aminergic neurotransmitters. Nature 1978, 274, 906– 908, DOI: 10.1038/274906a0[Crossref], [PubMed], [CAS], Google Scholar290https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1MXhsVKhtrs%253D&md5=85391a6eca50f7a14b59c0e9084573caSelective inhibitors of biosynthesis of aminergic neurotransmittersKollonitsch, J.; Patchett, A. A.; Marburg, S.; Maycock, A. L.; Perkins, L. M.; Doldouras, G. A.; Duggan, D. E.; Aster, S. D.Nature (London, United Kingdom) (1978), 274 (5674), 906-8CODEN: NATUAS; ISSN:0028-0836.α-Fluoromethylamino acids and α-fluoromethyl amines were irreversible inhibitors of the amino acid decarboxylases involved in the formation of aminergic neurotransmitters. The inhibition of dopa decarboxylase (EC 4.1.1.26) [9042-64-2] by (S)-α-fluoromethyldopa [69672-34-0] was retarded by α-methyldopa, suggesting that the active site was the site of inactivation. Inhibition seemed to involve the stoichiometric formation of an enzyme-inhibitor adduct lacking the CO2H and F- moiety of the inhibitor.291Maycock, A. L.; Aster, S. D.; Patchett, A. A. Inactivation of 3-(3,4-dihydroxyphenyl)alanine decarboxylase by 2-(fluoromethyl)-3-(3,4-dihydroxyphenyl)alanine. Biochemistry 1980, 19, 709– 718, DOI: 10.1021/bi00545a016[ACS Full Text], [CAS], Google Scholar291https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3cXhtVKgtLo%253D&md5=8740361411508e1e694cd03ef873685cInactivation of 3-(3,4-dihydroxyphenyl)alanine decarboxylase by 2-(fluoromethyl)-3-(3,4-dihydroxyphenyl)alanineMaycock, A. L.; Aster, S. D.; Patchett, A. A.Biochemistry (1980), 19 (4), 709-18CODEN: BICHAW; ISSN:0006-2960.2-(Fluoromethyl)-3-(3,4-dihydroxyphenyl)alanine (I) caused rapid, time-dependent, stereospecific, and irreversible inhibition of hog kidney arom. amino acid (DOPA) decarboxylase. The inactivation occurred with loss of both the carboxyl C and F- from I and resulted in the stoichiometric formation of a covalent enzyme-inhibitor adduct. The data are consistent with I being a suicide inactivator of the enzyme, and a plausible mechanism for the inactivation process is presented. The inactivation is highly efficient in that there is essentially no enzymic turnover of I to produce the corresponding amine, 1-(fluoromethyl)-2-(3,4-dihydroxyphenyl)ethylamine (II). II was also a potent inactivator of the enzyme. In vivo, I inactivated both brain and peripheral (liver) DOPA decarboxylase activity. The possible significance of these data with respect to the known antihypertensive effect of I is discussed.292Metcalf, B. W.; Bey, P.; Danzin, C.; Jung, M. J.; Casara, P.; Vevert, J. P. Catalytic irreversible inhibition of mammalian ornithine decarboxylase (E.C. 4.1.1.17) by substrate and product analogues. J. Am. Chem. Soc. 1978, 100, 2551– 2553, DOI: 10.1021/ja00476a050[ACS Full Text], [CAS], Google Scholar292https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXktF2jsbg%253D&md5=eb551e94129f2c9ccf9c0821756fd060Catalytic irreversible inhibition of mammalian ornithine decarboxylase (E.C.4.1.1.17) by substrate and product analogsMetcalf, B. W.; Bey, P.; Danzin, C.; Jung, M. J.; Casara, P.; Vevert, J. P.Journal of the American Chemical Society (1978), 100 (8), 2551-3CODEN: JACSAT; ISSN:0002-7863.Ornithine analogs incorporating an α-Me group functionalized by fluoro, chloro, and cyano substituents, as well as the putrescine analogs 5-hexyne-1,4-diamine (I) and trans-hex-2-ene-5-yne-1,4-diamine (II), were catalytic irreversible inhibitors of a prepn. of mammaliam ornithine decarboxylase. For the ornithine analogs, it is proposed that decarboxylation of the enzyme-bound Schiff's base formed between pyridoxal phosphate and the analog leads to a reactive imine which can alkylate a nucleophilic residue at the enzyme's active site. Enzyme inactivation by I and II is rationalized on the basis of the microscopic reversibility principle. It is proposed that the proton abstraction implicit in the reverse reaction leads to a reactive allene in the active site, which, once again, is an alkylating agent.293Qu, N.; Ignatenko, N. A.; Yamauchi, P.; Stringer, D. E.; Levenson, C.; Shannon, P.; Perrin, S.; Gerner, E. W. Inhibition of human ornithine decarboxylase activity by enantiomers of difluoromethylornithine. Biochem. J. 2003, 375, 465– 470, DOI: 10.1042/bj20030382[Crossref], [PubMed], [CAS], Google Scholar293https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXnvF2rsLo%253D&md5=21fae1a86ab5b589ae285f47b0278b6dInhibition of human ornithine decarboxylase activity by enantiomers of difluoromethylornithineQu, Ning; Ignatenko, Natalia A.; Yamauchi, Phillip; Stringer, David E.; Levenson, Corey; Shannon, Patrick; Perrin, Scott; Gerner, Eugene W.Biochemical Journal (2003), 375 (2), 465-470CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)Racemic difluoromethylornithine (D/L-DFMO) is an inhibitor of ODC (ornithine decarboxylase), the first enzyme in eukaryotic polyamine biosynthesis. D/L-DFMO is an effective anti-parasitic agent and inhibitor of mammalian cell growth and development. Purified human ODC-catalyzed ornithine decarboxylation is highly stereospecific. However, both DFMO enantiomers suppressed ODC activity in a time- and concn.-dependent manner. ODC activity failed to recover after treatment with either L- or D-DFMO and dialysis to remove free inhibitor. The inhibitor dissocn. const. (K D) values for the formation of enzyme-inhibitor complexes were 28.3 ± 3.4, 1.3 ± 0.3 and 2.2 ± 0.4 μM resp. for D-, L- and D/L-DFMO. The differences in these K D values were statistically significant (P <0.05). The inhibitor inactivation consts. (K inact) for the irreversible step were 0.25 ± 0.03, 0.15 ± 0.03 and 0.15 ± 0.03 min-1 resp. for D-, L- and D/L-DFMO. These latter values were not statistically significantly different (P >0.1). D-DFMO was a more potent inhibitor (IC50 ∼7.5 μM) when compared with D-ornithine (IC50 ∼1.5 mM) of ODC-catalyzed L-ornithine decarboxylation. Treatment of human colon tumor-derived HCT116 cells with either L- or D-DFMO decreased the cellular polyamine contents in a concn.-dependent manner. These results show that both enantiomers of DFMO irreversibly inactivate ODC and suggest that this inactivation occurs by a common mechanism. Both enantiomers form enzyme-inhibitor complexes with ODC, but the probability of formation of these complexes is 20 times greater for L-DFMO when compared with D-DFMO. The rate of the irreversible reaction in ODC inactivation is similar for the L- and D-enantiomer. This unexpected similarity between DFMO enantiomers, in contrast with the high degree of stereospecificity of the substrate ornithine, appears to be due to the α-substituent of the inhibitor. The D-enantiomer may have advantages, such as decreased normal tissue toxicity, over L- or D/L-DFMO in some clin. applications.294Wallace, H. M.; Fraser, A. V.; Hughes, A. A perspective of polyamine metabolism. Biochem. J. 2003, 376, 1– 14, DOI: 10.1042/bj20031327[Crossref], [PubMed], [CAS], Google Scholar294https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXovVajs7Y%253D&md5=106d3c7e36e100d304d1f1a6570db3e0A perspective of polyamine metabolismWallace, Heather M.; Fraser, Alison V.; Hughes, AlunBiochemical Journal (2003), 376 (1), 1-14CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)A review. Polyamines are essential for the growth and function of normal cells. They interact with various macromols., both electrostatically and covalently and, as a consequence, have a variety of cellular effects. The complexity of polyamine metab. and the multitude of compensatory mechanisms that are invoked to maintain polyamine homeostasis argue that these amines are crit. to cell survival. The regulation of polyamine content within cells occurs at several levels, including transcription and translation. In addn., novel features such as the +1 frameshift required for antizyme prodn. and the rapid turnover of several of the enzymes involved in the pathway make the regulation of polyamine metab. a fascinating subject. The link between polyamine content and human disease is unequivocal, and significant success has been obtained in the treatment of a no. of parasitic infections. Targeting the polyamine pathway as a means of treating cancer has met with limited success, although the development of drugs such as DFMO (α-difluoromethylornithine), a rationally designed anticancer agent, has revolutionized our understanding of polyamine function in cell growth and provided proof of concept' that influencing polyamine metab. and content within tumor cells will prevent tumor growth. The more recent development of the polyamine analogs has been pivotal in advancing our understanding of the necessity to deplete all three polyamines to induce apoptosis in tumor cells. The current thinking is that the polyamine inhibitors/analogs may also be useful agents in the chemoprevention of cancer and, in this area, we may yet see a revival of DFMO. The future will be in adopting a functional genomics approach to identifying polyamine-regulated genes linked to either carcinogenesis or apoptosis.295Casero, R. A., Jr.; Murray Stewart, T.; Pegg, A. E. Polyamine metabolism and cancer: treatments, challenges and opportunities. Nat. Rev. Cancer 2018, 18, 681– 695, DOI: 10.1038/s41568-018-0050-3[Crossref], [PubMed], [CAS], Google Scholar295https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1Gks7nF&md5=9bcfc74df8e65e0ce8a53203770bd1f1Polyamine metabolism and cancer: treatments, challenges and opportunitiesCasero, Jr, Robert A.; Murray Stewart, Tracy; Pegg, Anthony E.Nature Reviews Cancer (2018), 18 (11), 681-695CODEN: NRCAC4; ISSN:1474-175X. (Nature Research)Advances in our understanding of the metab. and mol. functions of polyamines and their alterations in cancer have led to resurgence in the interest of targeting polyamine metab. as an anticancer strategy. Increasing knowledge of the interplay between polyamine metab. and other cancer-driving pathways, including the PTEN-PI3K-mTOR complex 1 (mTORC1), WNT signalling and RAS pathways, suggests potential combination therapies that will have considerable clin. promise. Addnl., an expanding no. of promising clin. trials with agents targeting polyamines for both therapy and prevention are ongoing. New insights into mol. mechanisms linking dysregulated polyamine catabolism and carcinogenesis suggest addnl. strategies that can be used for cancer prevention in at-risk individuals. In addn., polyamine blocking therapy, a strategy that combines the inhibition of polyamine biosynthesis with the simultaneous blockade of polyamine transport, can be more effective than therapies based on polyamine depletion alone and may involve an antitumor immune response. These findings open up new avenues of research into exploiting aberrant polyamine metab. for anticancer therapy.296Meyskens, F. L., Jr.; Gerner, E. W. Development of difluoromethylornithine as a chemoprevention agent for the management of colon cancer. J. Cell. Biochem. 1995, 59 (S22), 126– 131, DOI: 10.1002/jcb.240590816297Levin, V. A.; Ictech, S. E.; Hess, K. R. Clinical importance of eflornithine (α-difluoromethylornithine) for the treatment of malignant gliomas. CNS Oncol. 2018, 7, CNS16, DOI: 10.2217/cns-2017-0031298Ferrins, L.; Rahmani, R.; Baell, J. B. Drug discovery and human African trypanosomiasis: a disease less neglected?. Future Med. Chem. 2013, 5, 1801– 1841, DOI: 10.4155/fmc.13.162[Crossref], [PubMed], [CAS], Google Scholar298https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1CitLzK&md5=89e0f13e2bd1f3bdb4821ba513754069Drug discovery and human African trypanosomiasis: a disease less neglected?Ferrins, Lori; Rahmani, Raphael; Baell, Jonathan B.Future Medicinal Chemistry (2013), 5 (15), 1801-1841CODEN: FMCUA7; ISSN:1756-8919. (Future Science Ltd.)A review. Human African trypanosomiasis (HAT) has been neglected for a long time. The most recent drug to treat this disease, eflornithine, was approved by the US FDA in 2000. Current treatments exhibit numerous problematic side effects and are often ineffective against the debilitating CNS resident stage of the disease. Fortunately, several partnerships and initiatives have been formed over the last 20 years in an effort to eradicate HAT, along with a no. of other neglected diseases. This has led to an increasing no. of foundations and research institutions that are currently working on the development of new drugs for HAT and tools with which to diagnose and treat patients. New biochem. pathways as therapeutic targets are emerging, accompanied by increasing nos. of new antitrypanosomal compd. classes. The future looks promising that this collaborative approach will facilitate eagerly awaited breakthroughs in the treatment of HAT.299LoGiudice, N.; Le, L.; Abuan, I.; Leizorek, Y.; Roberts, S. C. α-Difluoromethylornithine, an irreversible inhibitor of polyamine biosynthesis, as a therapeutic strategy against hyperproliferative and infectious diseases. Med. Sci. 2018, 6, 12, DOI: 10.3390/medsci6010012[Crossref], [CAS], Google Scholar299https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtVKktrfO&md5=3c525e3ef0ec516b797fa8a7e66dec15Alpha-difluoromethylornithine, an irreversible inhibitor of polyamine biosynthesis, as a therapeutic strategy against hyperproliferative and infectious diseasesLoGiudice, Nicole; Le, Linh; Abuan, Irene; Leizorek, Yvette; Roberts, Sigrid C.Medical Sciences (2018), 6 (1), 12/1-12/17CODEN: MSECFJ; ISSN:2076-3271. (MDPI AG)The fluorinated ornithine analog -difluoromethylornithine (DFMO, eflornithine, ornidyl) is an irreversible suicide inhibitor of ornithine decarboxylase (ODC), the first and rate-limiting enzyme of polyamine biosynthesis. The ubiquitous and essential polyamines have many functions, but are primarily important for rapidly proliferating cells. Thus, ODC is potentially a drug target for any disease state where rapid growth is a key process leading to pathol. The compd. was originally discovered as an anticancer drug, but its effectiveness was disappointing. However, DFMO was successfully developed to treat African sleeping sickness and is currently one of few clin. used drugs to combat this neglected tropical disease. The other Food and Drug Administration (FDA) approved application for DFMO is as an active ingredient in the hair removal cream Vaniqa. In recent years, renewed interest in DFMO for hyperproliferative diseases has led to increased research and promising preclin. and clin. trials. This review explores the use of DFMO for the treatment of African sleeping sickness and hirsutism, as well as its potential as a chemopreventive and chemotherapeutic agent against colorectal cancer and neuroblastoma.300Coyne, P. E., Jr. The eflornithine story. J. Am. Acad. Dermatol. 2001, 45, 784– 786, DOI: 10.1067/mjd.2001.117853[Crossref], [PubMed], [CAS], Google Scholar300https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3MrmtVOhtw%253D%253D&md5=374124f20a8698771d87bc950b3d54f7The eflornithine storyCoyne P E JrJournal of the American Academy of Dermatology (2001), 45 (5), 784-6 ISSN:0190-9622.There is no expanded citation for this reference.301McCune, C. D.; Beio, M. L.; Sturdivant, J. M.; de la Salud-Bea, R.; Darnell, B. M.; Berkowitz, D. B. Synthesis and deployment of an elusive fluorovinyl cation equivalent: Access to quaternary α-(1′-fluoro)vinyl amino acids as potential PLP enzyme inactivators. J. Am. Chem. Soc. 2017, 139, 14077– 14089, DOI: 10.1021/jacs.7b04690[ACS Full Text], [CAS], Google Scholar301https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsV2jsrnE&md5=1322ac88e9c11007dfb9d820a7a1d875Synthesis and deployment of an elusive fluorovinyl cation equivalent: Access to quaternary α-(1'-fluoro)vinyl amino acids as potential PLP enzyme inactivatorsMcCune, Christopher D.; Beio, Matthew L.; Sturdivant, Jill M.; de la Salud-Bea, Roberto; Darnell, Brendan M.; Berkowitz, David B.Journal of the American Chemical Society (2017), 139 (40), 14077-14089CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Developing specific chem. functionalities to deploy in biol. environments for targeted enzyme inactivation lies at the heart of mechanism-based inhibitor development but also is central to other protein-tagging methods in modern chem. biol. including activity-based protein profiling and proteolysis-targeting chimeras. We describe here a previously unknown class of potential PLP enzyme inactivators; namely, a family of quaternary, α-(1'-fluoro)vinyl amino acids, bearing the side chains of the cognate amino acids. These are obtained by the capture of suitably protected amino acid enolates with β,β-difluorovinyl Ph sulfone, a new (1'-fluoro)vinyl cation equiv., and an electrophile that previously eluded synthesis, capture and characterization. A significant variety of biol. relevant AA side chains are tolerated including those for alanine, valine, leucine, methionine, lysine, phenylalanine, tyrosine, and tryptophan. Following addn./elimination, the resulting transoid α-(1'-fluoro)-β-(phenylsulfonyl)vinyl AA-esters undergo smooth sulfone-stannane interchange to stereoselectively give the corresponding transoid α-(1'-fluoro)-β-(tributylstannyl)vinyl AA-esters. Protodestannylation and global deprotection then yield these sterically encumbered and densely functionalized quaternary amino acids. The α-(1'-fluoro)vinyl trigger, a potential allene-generating functionality originally proposed by Abeles, is now available in a quaternary AA context for the first time. In an initial test of this new inhibitor class, α-(1'-fluoro)vinyllysine is seen to act as a time-dependent, irreversible inactivator of lysine decarboxylase from Hafnia alvei. The enantiomers of the inhibitor could be resolved, and each is seen to give time-dependent inactivation with this enzyme. Kitz-Wilson anal. reveals similar inactivation parameters for the two antipodes, L-α-(1'-fluoro)vinyllysine (Ki = 630 ± 20 μM; t1/2 = 2.8 min) and D-α-(1'-fluoro)vinyllysine (Ki = 470 ± 30 μM; t1/2 = 3.6 min). The stage is now set for exploration of the efficacy of this trigger in other PLP-enzyme active sites.302Abeles, R. H.; Alston, T. A. Enzyme inhibition by fluoro compounds. J. Biol. Chem. 1990, 265, 16705– 16708[PubMed], [CAS], Google Scholar302https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXisVKh&md5=9b91b5367f5c8085a9b00e90d0e81d37Enzyme inhibition by fluoro compoundsAbeles, Robert H.; Alston, Theodore A.Journal of Biological Chemistry (1990), 265 (28), 16705-8CODEN: JBCHA3; ISSN:0021-9258.A review, with 91 refs., on F in enzyme inhibitors (including drugs and toxins). F properties (i.e. steric compactness and electronegativity) are considered with regard to replacement of H and OH groups, inductive effects, and function without F as a leaving group.303Xu, Y.; Abeles, R. H. Inhibition of tryptophan synthase by (1-fluorovinyl)glycine. Biochemistry 1993, 32, 806– 811, DOI: 10.1021/bi00054a010[ACS Full Text], [CAS], Google Scholar303https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXlsV2hsg%253D%253D&md5=502800786642e33ed3f4d5fa1b97ef30Inhibition of tryptophan synthase by (1-fluorovinyl)glycineXu, Yajun; Abeles, Robert H.Biochemistry (1993), 32 (3), 806-11CODEN: BICHAW; ISSN:0006-2960.Tryptophan synthase (α2β2 complex) from Salmonella typhimurium catalyzes the formation of tryptophan from serine and indole. The enzyme is inactivated by (1-fluorovinyl)glycine. Concomitant with enzyme inactivation, the absorbance at 485 nm increases, indicating covalent modification of pyridoxal 5'-phosphate. It is proposed that inactivation involves elimination of HF to form an allene, which reacts with a nucleophile at the active site. The inactivation reaction involves an α,β-elimination, as does the formation of tryptophan from indole and serine. The inactivation occurs with kin > 1.3 s-1, which is very close to kcat (6.4 s-1) for the formation of tryptophan from indole and serine. The inactive enzyme (α2β2) regains activity with koff = 0.005 min-1. Aminoacetone is formed during reactivation, and pyridoxal 5'-phosphate is regenerated. Tryptophan synthase also catalyzes the dehydration of serine, or 3-fluoroalanine, to pyruvate in the absence of indole. This reaction involves an α,β-elimination and the intermediate formation of an aminoacrylate adduct with pyridoxal 5'-phosphate, as does the formation of tryptophan. Pyruvate formation proceeds at less than 5% the rate of tryptophan formation. With [2-2H]serine an isotope effect (DVmax = 1.5) is obsd. It is proposed that pyruvate formation is limited by the rate of hydration of the aminoacrylate intermediate and the rate of the abstraction of the serine α-hydrogen.304Silverman, R. B.; Levy, M. A. Substituted 4-aminobutanoic acids. Substrates for γ-aminobutyric acid α-ketoglutaric acid aminotransferase. J. Biol. Chem. 1981, 256, 11565– 11568[PubMed], [CAS], Google Scholar304https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXmtFKqtLw%253D&md5=c85d3103bf565c075232641eebacc8feSubstituted 4-aminobutanoic acids. Substrates for γ-aminobutyric acid α-ketoglutaric acid aminotransferaseSilverman, Richard B.; Levy, Mark A.Journal of Biological Chemistry (1981), 256 (22), 11565-8CODEN: JBCHA3; ISSN:0021-9258.Substituted 4-aminobutanoic acids were studied as potential irreversible inactivators of purified pig brain γ-aminobutyrate (GABA) transaminase, the enzyme responsible for the degrdn. of the inhibitory neurotransmitter, GABA. Unlike the related 4-amino-5-halopentanoic acids, the 4-amino-3-halobutanoic acids are substrates for this enzyme, undergoing exclusive elimination to succinic semialdehyde and producing no inactivation. The hydroxy analog, however, undergoes exclusive transamination and no succinic semialdehyde is detected. These results are discussed in terms of the nature of the substituents, the structure of the active site of GABA transaminase, and the design of mechanism-based inactivators.305Clift, M. D.; Ji, H.; Deniau, G. P.; O’Hagan, D.; Silverman, R. B. Enantiomers of 4-amino-3-fluorobutanoic acid as substrates for γ-aminobutyric acid aminotransferase. Conformational probes for GABA binding. Biochemistry 2007, 46, 13819– 13828, DOI: 10.1021/bi701249q[ACS Full Text], [CAS], Google Scholar305https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1OhsbrO&md5=40d7bf78aab98245d752399c4f468bf1Enantiomers of 4-Amino-3-fluorobutanoic Acid as Substrates for γ-Aminobutyric Acid Aminotransferase. Conformational Probes for GABA BindingClift, Michael D.; Ji, Haitao; Deniau, Gildas P.; O'Hagan, David; Silverman, Richard B.Biochemistry (2007), 46 (48), 13819-13828CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)γ-Aminobutyric acid aminotransferase (GABA-AT), a pyridoxal 5'-phosphate dependent enzyme, catalyzes the degrdn. of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) to succinic semialdehyde with concomitant conversion of pyridoxal 5'-phosphate (PLP) to pyridoxamine 5'-phosphate (PMP). The enzyme then catalyzes the conversion of α-ketoglutarate to the excitatory neurotransmitter L-glutamate. Racemic 4-amino-3-fluorobutanoic acid (3-F-GABA) was shown previously to act as a substrate for GABA-AT, not for transamination, but for HF elimination. Here we report studies of the reaction catalyzed by GABA-AT on (R)- and (S)-3-F-GABA. Neither enantiomer is a substrate for transamination. Very little elimination from the (S)-enantiomer was detected using a coupled enzyme assay; The rate of elimination of HF from the (R)-enantiomer is at least 10 times greater than that for the (S)-enantiomer. The (R)-enantiomer is about 20 times more efficient as a substrate for GABA-AT catalyzed HF elimination than GABA is a substrate for transamination. The (R)-enantiomer also inhibits the transamination of GABA 10 times more effectively than the (S)-enantiomer. Using a combination of computer modeling and the knowledge that vicinal C-F and C-NH3+ bonds have a strong preference to align gauche rather than anti to each other, it is concluded that on binding of free 3-F-GABA to GABA-AT the optimal conformation places the C-NH3+ and C-F bonds gauche in the (R)-enantiomer but anti in the (S)-enantiomer. Furthermore, the dynamic binding process and the bioactive conformation of GABA bound to GABA-AT have been inferred on the basis of the different biol. behavior of the two enantiomers of 3-F-GABA when they bind to the enzyme. The present study suggests that the C-F bond can be utilized as a conformational probe to explore the dynamic binding process and provide insight into the bioactive conformation of substrates, which cannot be easily detd. by other biophys. approaches.306Deniau, G.; Slawin, A. M.; Lebl, T.; Chorki, F.; Issberner, J. P.; van Mourik, T.; Heygate, J. M.; Lambert, J. J.; Etherington, L. A.; Sillar, K. T.; O’Hagan, D. Synthesis, conformation and biological evaluation of the enantiomers of 3-fluoro-γ-aminobutyric acid ((R)- and (S)-3F-GABA): an analogue of the neurotransmitter GABA. ChemBioChem 2007, 8, 2265– 2274, DOI: 10.1002/cbic.200700371[Crossref], [PubMed], [CAS], Google Scholar306https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXltlentbc%253D&md5=9ebd7a5dc80ad3dd9c711572f1304f32Synthesis, conformation and biological evaluation of the enantiomers of 3-fluoro-γ-aminobutyric acid ((R)- and (S)-3F-GABA): an analog of the neurotransmitter GABADeniau, Gildas; Slawin, Alexandra M. Z.; Lebl, Tomas; Chorki, Fatima; Issberner, Jon P.; van Mourik, Tanja; Heygate, Judith M.; Lambert, Jeremy J.; Etherington, Lori-An; Sillar, Keith T.; O'Hagan, DavidChemBioChem (2007), 8 (18), 2265-2274CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)β-Aminobutyric acid or GABA (1) is one of the major inhibitory amino acid neurotransmitters of the central nervous system. This article describes the first synthesis of both the (R)- and (S)- enantiomers of 3-fluoro-GABA (2, 3F-GABA). DFT calcns. were carried out in a continuum solvent model (PCM-B3LYP) to est. the preferred conformations of 3F-GABA in aq. soln. NMR coupling consts. were calcd. for each conformer and were then used to simulate the NMR spectra to evaluate the soln. conformation of 3F-GABA. A preliminary evaluation of the 3F-GABA enantiomers shows that they act similarly as agonists of cloned GABAA receptors; however, they behave quite differently in a whole animal (Xenopus laevis tadpole model).307Buissonneaud, D. Y.; van Mourik, T.; O’Hagan, D. A DFT study on the origin of the fluorine gauche effect in substituted fluoroethanes. Tetrahedron 2010, 66, 2196– 2202, DOI: 10.1016/j.tet.2010.01.049[Crossref], [CAS], Google Scholar307https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXitleiurk%253D&md5=b7d6e3599c8e0b9a6364f55c34f362afA DFT study on the origin of the fluorine gauche effect in substituted fluoroethanesBuissonneaud, David Y.; van Mourik, Tanja; O'Hagan, DavidTetrahedron (2010), 66 (12), 2196-2202CODEN: TETRAB; ISSN:0040-4020. (Elsevier Ltd.)DFT derived conformational energy profiles of a series of β-substituted α-fluoroethanes (F-CH2CH2-X) have been explored where the substituent X was varied as NH3+, OCOH, NCO, NO2, NHCHO, F, N3, CH=NH, NCS, CH=C=CH2, CH3, CH=CH2, NC, CN, CHO, and CCH. Comparisons were correlated relative to 1,2-difluoroethane, a compd. which exhibits a well known gauche preference. Only four of the compds. displayed an anti preference, with the large majority preferring a gauche conformation. In particular the influence of steric and electrostatic attraction/repulsion between the fluorine atom and the X-substituent was explored by evaluating rotational energy profiles for all compds. and sep. NBO correlations were evaluated to assess the contribution of hyperconjugation to the minimized gauche and anti conformers. In the event the gauche preference for 1,2-difluoroethane was shown to have an origin due largely to σ(C-H)→σ*(C-F) hyperconjugative interactions, whereas the conformational preference for the remaining structures is rationalized by hyperconjugative as well as steric and electrostatic contributions. The anti preferred compds. 13, 14 and 16 possessed triple bonds and the preference arose due to fluorine/p-orbital repulsion.308Boeckxstaens, G. E.; Denison, H.; Jensen, J. M.; Lehmann, A.; Ruth, M. Translational gastrointestinal pharmacology in the 21st century: ’the lesogaberan story. Curr. Opin. Pharmacol. 2011, 11, 630– 633, DOI: 10.1016/j.coph.2011.10.011[Crossref], [PubMed], [CAS], Google Scholar308https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFegsbbF&md5=8e8fb9878270ac3f1d68bd6ad2b2b4deTranslational gastrointestinal pharmacology in the 21st century: the lesogaberan story'Boeckxstaens, Guy E.; Denison, Hans; Jensen, Joergen M.; Lehmann, Anders; Ruth, MagnusCurrent Opinion in Pharmacology (2011), 11 (6), 630-633CODEN: COPUBK; ISSN:1471-4892. (Elsevier Ltd.)A review. The development of the novel γ-aminobutyric acid type-B receptor (GABAB) agonist lesogaberan is presented as an example of a partly successful translational strategy in the field of gastroenterol. Data on transient lower esophageal sphincter relaxations (TLESRs) and gastroesophageal reflux inhibition from preclin. models translated well to clin. studies in healthy volunteers and patients with gastroesophageal reflux disease (GERD). Animal models have also been used successfully to predict the effect of other target mechanisms on TLESRs in humans. However, while translation of physiol. to symptomatol. in patients with GERD was achieved, the effect size was too small to be of clin. significance. A deeper understanding of the cause of symptoms in different patient categories is therefore required.309Ekdahl, A.; Aurell-Holmberg, A.; Castagnoli, N., Jr. Identification of the metabolites of lesogaberan using linear trap quadrupole orbitrap mass spectrometry and hydrophilic interaction liquid chromatography. Xenobiotica 2013, 43, 461– 467, DOI: 10.3109/00498254.2012.725486[Crossref], [PubMed], [CAS], Google Scholar309https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXltVOqsrk%253D&md5=bf71cca5a686d1bee441e1ce842647caIdentification of the metabolites of lesogaberan using linear trap quadrupole orbitrap mass spectrometry and hydrophilic interaction liquid chromatographyEkdahl, Anja; Aurell-Holmberg, Ann; Castagnoli, Neal, Jr.Xenobiotica (2013), 43 (5), 461-467CODEN: XENOBH; ISSN:0049-8254. (Informa Healthcare)In this study, hydrophilic interaction liq. chromatog. (HILIC), radiochem. activity monitoring and linear trap quadrupole orbitrap mass spectrometry (MS) and tandem mass spectrometry (MS/MS) were used to identify the metabolites of a highly polar novel γ-aminobutyric acid type-B receptor agonist, lesogaberan, in rats. Urine was collected from 3 male Wistar rats for 24 h after dosing with 14C-labeled lesogaberan (170 mg/kg, 10 MBq/kg); blood plasma samples were taken 2 and 24 h after dosing. Pooled samples were sepd. by HILIC and eluents were analyzed by radiochem. activity monitoring, MS and MS/MS. Only the parent compd. was detected in plasma, but 6 metabolites (M1-M6) were detected in urine. Anal. of MS and MS/MS data and comparison with synthetic ref. stds. enabled the identification of the structure of each metabolite. M1 was identified as the N-acetylated species [(2R)-3-acetamido-2-fluoropropyl]-phosphinic acid, and M6 as [(2R)-3-amino-2-fluoropropyl]-phosphonic acid. Metabolites M2 and M5 were the alc. and carboxylic acid species 3-hydroxypropyl-phosphinic acid and 3-hydroxyphosphonoyl-propanoic acid, resp., both of which had lost the fluorine atom present in the parent compd. M3 was the corresponding carboxylic acid species retaining the fluorine atom, (2R)-2-fluoro-3-hydroxyphosphonoyl-propanoic acid. Finally M4 was identified as [(2R)-2-fluoro-3-guanidino-propyl]-phosphinic acid.310Lehmann, A.; Antonsson, M.; Holmberg, A. A.; Blackshaw, L. A.; Branden, L.; Brauner-Osborne, H.; Christiansen, B.; Dent, J.; Elebring, T.; Jacobson, B. M.; Jensen, J.; Mattsson, J. P.; Nilsson, K.; Oja, S. S.; Page, A. J.; Saransaari, P.; von Unge, S. R)-(3-amino-2-fluoropropyl) phosphinic acid (AZD3355), a novel GABAB receptor agonist, inhibits transient lower esophageal sphincter relaxation through a peripheral mode of action. J. Pharmacol. Exp. Ther. 2009, 331, 504– 512, DOI: 10.1124/jpet.109.153593[Crossref], [PubMed], [CAS], Google Scholar310https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlKnu7zI&md5=3f342dd2efa576ec5b3362ece793594f(R)-(3-amino-2-fluoropropyl) phosphinic acid (AZD3355), a novel GABAB receptor agonist, inhibits transient lower esophageal sphincter relaxation through a peripheral mode of actionLehmann, Anders; Antonsson, Madeleine; Holmberg, Ann Aurell; Blackshaw, L. Ashley; Braenden, Lena; Braeuner-Osborne, Hans; Christiansen, Bolette; Dent, John; Elebring, Thomas; Jacobson, Britt-Marie; Jensen, Joergen; Mattsson, Jan P.; Nilsson, Karolina; Oja, Simo S.; Page, Amanda J.; Saransaari, Pirjo; von Unge, SverkerJournal of Pharmacology and Experimental Therapeutics (2009), 331 (2), 504-512CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)Gastroesophageal reflux disease (GERD) affects >10% of the Western population. Conventionally, GERD is treated by reducing gastric acid secretion, which is effective in most patients but inadequate in a significant minority. We describe a new therapeutic approach for GERD, based on inhibition of transient lower esophageal sphincter relaxation (TLESR) with a proposed peripherally acting GABAB receptor agonist, (R)-(3-amino-2-fluoropropyl)phosphinic acid (AZD3355). AZD3355 potently stimulated recombinant human GABAB receptors and inhibited TLESR in dogs, with a biphasic dose-response curve. In mice, AZD3355 produced considerably less central side effects than the prototypical GABAB receptor agonist baclofen but evoked hypothermia at very high doses (blocked by a GABAB receptor antagonist and absent in GABAB-/- mice). AZD3355 and baclofen differed markedly in their distribution in rat brain; AZD3355, but not baclofen, was concd. in circumventricular organs as a result of active uptake (shown by avid intracellular sequestration) and related to binding of AZD3355 to native GABA transporters in rat cerebrocortical membranes. AZD3355 was also shown to be transported by all four recombinant human GABA transporters. AR-H061719 [(R/S)-(3-amino-2-fluoropropyl)phosphinic acid], (the racemate of AZD3355) inhibited the response of ferret mechanoreceptors to gastric distension, further supporting its peripheral site of action on TLESR. In summary, AZD3355 probably inhibits TLESR through stimulation of peripheral GABAB receptors and may offer a potential new approach to treatment of GERD.311Darland, G. K.; Hajdu, R.; Kropp, H.; Kahan, F. M.; Walker, R. W.; Vandenheuvel, W. J. Oxidative and defluorinative metabolism of fludalanine, 2-2H-3-fluoro-d-alanine. Drug Metab. Dispos. 1986, 14, 668– 673[PubMed], [CAS], Google Scholar311https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXnvVWltA%253D%253D&md5=554409be533e8ce74036e9ca2cf19dfbOxidative and defluorinative metabolism of fludalanine, 2-2H-3-fluoro-D-alanineDarland, Gary K.; Hajdu, Richard; Kropp, Helmut; Kahan, Frederick M.; Walker, Robert W.; Vandenheuvel, William J. A.Drug Metabolism and Disposition (1986), 14 (6), 668-73CODEN: DMDSAI; ISSN:0090-9556.The antibacterial agent fludalanine [35523-45-6] (used in this study as the deuterated analog 2-2H-3-fluoro-D-alanine (DFA) [35523-45-6]) is a potent inhibitor of bacterial alanine racemase, an enzyme required for the generation of D-alanine, an essential component of the bacterial cell wall. Primary metab. of DFA involves its oxidn. to fluoropyruvate (FP) [433-48-7]; this org. F- is then rapidly reduced to fluorolactate (FL) [433-47-6], which is the major org. metabolite in lab. animals. Gas-liq. chromatog. chem. ionization mass spectrometric assays were developed for these 2 metabolites. FL is the predominant organofluoride metabolite of DFA in the circulation. FP was detected in the urine although recovery was very low. The rapid conversion of FP to FL precludes assay of the former in serum. Max. serum FL concns. in the rat appear about 1 h after the dose of DFA and are relatively const. for several hours thereafter. The peak FL concn. is proportional to the dose of DFA; repeated daily dosing of DFA appears to cause neither satn. nor induction of metabolic pathways. Comparison of FL concns. detd. using the GC/MS assay with those based on an enzymic method specific for L-(+)-FL [3130-92-5] demonstrated that only the latter isomer is found in the plasma of monkeys dosed with DFA. In vivo exchange studies involving the α-proton of FL indicate that a small FP pool exists and is in equil. with FL. A crude pyruvate dehydrogenase [9014-20-4] complex isolated from beef heart mitochondria was shown to produce equimolar quantities of acetate [64-19-7], CO2, and F- from FP.312Patel, H.; Nemeria, N. S.; Andrews, F. H.; McLeish, M. J.; Jordan, F. Identification of charge transfer transitions related to thiamin-bound intermediates on enzymes provides a plethora of signatures useful in mechanistic studies. Biochemistry 2014, 53, 2145– 2152, DOI: 10.1021/bi4015743[ACS Full Text], [CAS], Google Scholar312https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXktlSrt70%253D&md5=4c355daabd03dde83b9ae902d7a39844Identification of charge transfer transitions related to thiamin-bound intermediates on enzymes provides a plethora of signatures useful in mechanistic studiesPatel, Hetalben; Nemeria, Natalia S.; Andrews, Forest H.; McLeish, Michael J.; Jordan, FrankBiochemistry (2014), 53 (13), 2145-2152CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The identification of enzyme-bound intermediates via their spectroscopic signatures, which then allows direct monitoring of the kinetic fate of these intermediates, poses a continuing challenge. As an electrophilic covalent catalyst, the thiamin diphosphate (ThDP) coenzyme forms a no. of noncovalent and covalent intermediates along its reaction pathways, and multiple UV-visible and CD spectroscopic bands have been identified by the authors pertinent to several among them. These electronic transitions fall into 2 classes: those for which the conjugated system provides a reasonable guide to the obsd. λmax and others in which there is no corresponding conjugated system and the obsd. CD bands are best ascribed to charge transfer (CT) transitions. Here, the authors report the reaction of 4 ThDP-dependent enzymes (pyruvate decarboxylase, pyruvate and oxoglutarate dehydrogenase complex E1 components, and benzaldehyde lyase) with alternate substrates: (a) acetylpyruvate, its Me ester, and fluoropyruvate, these providing the shortest side-chains attached at the thiazolium C2 atom and leading to CT bands with λmax values of >390 nm, not pertinent to any on-pathway conjugated systems (estd. λmax values of <330 nm), and (b) (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid displaying both a conjugated enamine (430 nm) and a CT transition (480 nm). The authors suggest that the CT transitions result from an interaction of the π bond on the ThDP C2 side-chain as a donor, and the pos. charged thiazolium ring as an acceptor, and correspond to covalent ThDP-bound intermediates. Time resoln. of these bands allows the rate consts. for individual steps to be detd. These CD methods can be applied to the entire ThDP superfamily of enzymes and should find applications with other enzymes.313Cordes, E. E. Chapter 11. Fludalanine. Nice try but no hallelujah. In Hallelujah Moments: Tales of Drug Discovery; Oxford University Press: New York, 2014; pp 183– 196.314Zhang, D.; Ogan, M.; Gedamke, R.; Roongta, V.; Dai, R.; Zhu, M.; Rinehart, J. K.; Klunk, L.; Mitroka, J. Protein covalent binding of Maxipost through a cytochrome P450-mediated ortho-quinone methide intermediate in rats. Drug Metab. Dispos. 2003, 31, 837– 845, DOI: 10.1124/dmd.31.7.837[Crossref], [PubMed], [CAS], Google Scholar314https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXltFalu7s%253D&md5=db2e7b4ad296af0226ac6d8b6b7b37adProtein covalent binding of maxipost through a cytochrome P450-mediated ortho-quinone methide intermediate in ratsZhang, Donglu; Ogan, Marc; Gedamke, Richard; Roongta, Vikram; Dai, Renke; Zhu, Mingshe; Rinehart, J. Kent; Klunk, Lewis; Mitroka, JamesDrug Metabolism and Disposition (2003), 31 (7), 837-845CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)(3S)-(+)-(5-Chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-((trifluoromethyl)-2H-indole-2-one) (MaxiPost, BMS-204352) is a potent and specific opener for maxi-K channels and has potential to prevent and treat ischemic stroke. Following single i.v. doses of [14C]BMS-204352 to rats, only 10 to 12% of radioactivity was extractable from plasma with org. solvents. The unextractable radioactivity remained assocd. with the proteins (mostly albumin) after SDS-polyacrylamide gel electrophoresis or dialysis. Following acid hydrolysis in 6 M HCl for 24 h at 110° from plasma proteins collected from nine rats dosed with [14C]BMS-204352, one major radioactive product was isolated and identified as a lysine-adduct of des-fluoro des-O-Me BMS-204352 by liq. chromatog./mass spectrometry and NMR analyses as well as by comparison with the synthetic analog, lysine-adduct of des-fluoro BMS-204352 (BMS-349821). The covalent binding of BMS-204352 results from the displacement of the ring-fluorine atom of des-O-Me BMS-204352 with the ε-amino group of a lysine residue. Microsomal incubations of [14C]BMS-204352 resulted in low levels of covalent binding of radioactivity to proteins. This in vitro covalent binding required cytochrome P 450-reductase cofactor NADPH and was attenuated by glutathione. P 4503A inhibitors ketoconazole and troleadomycin selectively prevented the covalent binding in vitro. Based on these observations, a two-step bioactivation process for the protein covalent binding of BMS-204352 was postulated:. (1) P 4503A-mediated O-demethylation leading to spontaneous release of HF and the formation of an ortho-quinone methide reactive metabolite and. (2) Nucleophilic addn. of the ε-amino group of protein lysine residue(s) in protein to form des-fluoro des-O-Me BMS-204352 lysine adduct.315Hewawasam, P.; Gribkoff, V. K.; Pendri, Y.; Dworetzky, S. I.; Meanwell, N. A.; Martinez, E.; Boissard, C. G.; Post-Munson, D. J.; Trojnacki, J. T.; Yeleswaram, K.; Pajor, L. M.; Knipe, J.; Gao, Q.; Perrone, R.; Starrett, J. E., Jr. The synthesis and characterization of BMS-204352 (MaxiPost) and related 3-fluorooxindoles as openers of maxi-K potassium channels. Bioorg. Med. Chem. Lett. 2002, 12, 1023– 1026, DOI: 10.1016/S0960-894X(02)00101-4[Crossref], [PubMed], [CAS], Google Scholar315https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XitFWhsLw%253D&md5=445a2b24bc60ad13a70a421d201a91d9The synthesis and characterization of BMS-204352 (MaxiPost) and related 3-fluorooxindoles as openers of maxi-K potassium channelsHewawasam, Piyasena; Gribkoff, Valentin K.; Pendri, Yadagiri; Dworetzky, Steven I.; Meanwell, Nicholas A.; Martinez, Eduardo; Boissard, Christopher G.; Post-Munson, Debra J.; Trojnacki, Joanne T.; Yeleswaram, Krishnaswamy; Pajor, Lorraine M.; Knipe, Jay; Gao, Qi; Perrone, Robert; Starrett, John E.Bioorganic & Medicinal Chemistry Letters (2002), 12 (7), 1023-1026CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science Ltd.)3-Aryl-3-fluorooxindoles can be efficiently synthesized in two steps by the addn. of an aryl Grignard to an isatin, followed by treatment with DAST. Oxindole 1 (BMS-204352; MaxiPost) can be isolated using chiral HPLC or prepd. by employing chiral resoln. Cloned maxi-K channels are opened by 1, which demonstrates a brain/plasma ratio >9 in rats.316Lee, K. L.; Ambler, C. M.; Anderson, D. R.; Boscoe, B. P.; Bree, A. G.; Brodfuehrer, J. I.; Chang, J. S.; Choi, C.; Chung, S.; Curran, K. J.; Day, J. E.; Dehnhardt, C. M.; Dower, K.; Drozda, S. E.; Frisbie, R. K.; Gavrin, L. K.; Goldberg, J. A.; Han, S.; Hegen, M.; Hepworth, D.; Hope, H. R.; Kamtekar, S.; Kilty, I. C.; Lee, A.; Lin, L. L.; Lovering, F. E.; Lowe, M. D.; Mathias, J. P.; Morgan, H. M.; Murphy, E. A.; Papaioannou, N.; Patny, A.; Pierce, B. S.; Rao, V. R.; Saiah, E.; Samardjiev, I. J.; Samas, B. M.; Shen, M. W. H.; Shin, J. H.; Soutter, H. H.; Strohbach, J. W.; Symanowicz, P. T.; Thomason, J. R.; Trzupek, J. D.; Vargas, R.; Vincent, F.; Yan, J.; Zapf, C. W.; Wright, S. W. Discovery of clinical candidate 1-{[(2S,3S,4S)-3-ethyl-4-fluoro-5-oxopyrrolidin-2-yl]methoxy}-7-methoxyisoquinoli ne-6-carboxamide (PF-06650833), a potent, selective inhibitor of interleukin-1 receptor associated kinase 4 (IRAK4), by fragment-based drug design. J. Med. Chem. 2017, 60, 5521– 5542, DOI: 10.1021/acs.jmedchem.7b00231[ACS Full Text], [CAS], Google Scholar316https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXnslGqu7c%253D&md5=9771ad2d33089f6ef3dba3556536ff1aDiscovery of Clinical Candidate 1-{[(2S,3S,4S)-3-Ethyl-4-fluoro-5-oxopyrrolidin-2-yl]methoxy}-7-methoxyisoquinoline-6-carboxamide (PF-06650833), a Potent, Selective Inhibitor of Interleukin-1 Receptor Associated Kinase 4 (IRAK4), by Fragment-Based Drug DesignLee, Katherine L.; Ambler, Catherine M.; Anderson, David R.; Boscoe, Brian P.; Bree, Andrea G.; Brodfuehrer, Joanne I.; Chang, Jeanne S.; Choi, Chulho; Chung, Seungwon; Curran, Kevin J.; Day, Jacqueline E.; Dehnhardt, Christoph M.; Dower, Ken; Drozda, Susan E.; Frisbie, Richard K.; Gavrin, Lori K.; Goldberg, Joel A.; Han, Seungil; Hegen, Martin; Hepworth, David; Hope, Heidi R.; Kamtekar, Satwik; Kilty, Iain C.; Lee, Arthur; Lin, Lih-Ling; Lovering, Frank E.; Lowe, Michael D.; Mathias, John P.; Morgan, Heidi M.; Murphy, Elizabeth A.; Papaioannou, Nikolaos; Patny, Akshay; Pierce, Betsy S.; Rao, Vikram R.; Saiah, Eddine; Samardjiev, Ivan J.; Samas, Brian M.; Shen, Marina W. H.; Shin, Julia H.; Soutter, Holly H.; Strohbach, Joseph W.; Symanowicz, Peter T.; Thomason, Jennifer R.; Trzupek, John D.; Vargas, Richard; Vincent, Fabien; Yan, Jiangli; Zapf, Christoph W.; Wright, Stephen W.Journal of Medicinal Chemistry (2017), 60 (13), 5521-5542CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Through fragment-based drug design focused on engaging the active site of IRAK4 and leveraging three-dimensional topol. in a ligand-efficient manner, a micromolar hit identified from a screen of a Pfizer fragment library was optimized to afford IRAK4 inhibitors with nanomolar potency in cellular assays. The medicinal chem. effort featured the judicious placement of lipophilicity, informed by cocrystal structures with IRAK4 and optimization of ADME properties to deliver clin. candidate I. This compd. benefitted from a 5-unit increase in lipophilic efficiency from the fragment hit, excellent kinase selectivity, and pharmacokinetic properties suitable for oral administration.317Dossetter, A. G. A statistical analysis of in vitro human microsomal metabolic stability of small phenyl group substituents, leading to improved design sets for parallel SAR exploration of a chemical series. Bioorg. Med. Chem. 2010, 18, 4405– 4414, DOI: 10.1016/j.bmc.2010.04.077[Crossref], [PubMed], [CAS], Google Scholar317https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXntV2rurc%253D&md5=93c9825d5da0b47e0f4c66996c883642A statistical analysis of in vitro human microsomal metabolic stability of small phenyl group substituents, leading to improved design sets for parallel SAR exploration of a chemical seriesDossetter, Alexander G.Bioorganic & Medicinal Chemistry (2010), 18 (12), 4405-4414CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)An anal. of in vitro human liver microsomal turnover assay results from a large dataset (∼75 K) of exptl. compds. tested is presented. Combined with an anal. of small (<6 Ha) substituents on known drugs and existing published results a new set of 29 substituents (consensus) is proposed to increase stability and probe SAR (an enhanced Topliss set'). In addn. a different group of 28 substituents are identified as unlikely to change in vitro HLM stability, and a further set of compds. focuses on increasing HLM stability only.318Lin, L. S.; Lanza, T. J., Jr.; Jewell, J. P.; Liu, P.; Shah, S. K.; Qi, H.; Tong, X.; Wang, J.; Xu, S. S.; Fong, T. M.; Shen, C. P.; Lao, J.; Xiao, J. C.; Shearman, L. P.; Stribling, D. S.; Rosko, K.; Strack, A.; Marsh, D. J.; Feng, Y.; Kumar, S.; Samuel, K.; Yin, W.; Van der Ploeg, L. H.; Goulet, M. T.; Hagmann, W. K. Discovery of N-[(1S,2S)-3-(4-Chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364), a novel, acyclic cannabinoid-1 receptor inverse agonist for the treatment of obesity. J. Med. Chem. 2006, 49, 7584– 7587, DOI: 10.1021/jm060996+[ACS Full Text], [CAS], Google Scholar318https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlCqsLvK&md5=e016bb35e491de4422bef3f3fa87ae84Discovery of N-[(1S,2S)-3-(4-Chlorophenyl)-2- (3-cyanophenyl)-1-methylpropyl]-2-methyl-2- {[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364), a Novel, Acyclic Cannabinoid-1 Receptor Inverse Agonist for the Treatment of ObesityLin, Linus S.; Lanza, Thomas J., Jr.; Jewell, James P.; Liu, Ping; Shah, Shrenik K.; Qi, Hongbo; Tong, Xinchun; Wang, Junying; Xu, Suoyu S.; Fong, Tung M.; Shen, Chun-Pyn; Lao, Julie; Xiao, Jing Chen; Shearman, Lauren P.; Stribling, D. Sloan; Rosko, Kimberly; Strack, Alison; Marsh, Donald J.; Feng, Yue; Kumar, Sanjeev; Samuel, Koppara; Yin, Wenji; Van der Ploeg, Lex H. T.; Goulet, Mark T.; Hagmann, William K.Journal of Medicinal Chemistry (2006), 49 (26), 7584-7587CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The discovery of novel acyclic amide cannabinoid-1 receptor inverse agonists is described. They are potent, selective, orally bioavailable, and active in rodent models of food intake and body wt. redn. A major focus of the optimization process was to increase in vivo efficacy and to reduce the potential for formation of reactive metabolites. These efforts led to the identification of compd. 48 (I)for development as a clin. candidate for the treatment of obesity.319Elliott, E. C.; Regan, S. L.; Maggs, J. L.; Bowkett, E. R.; Parry, L. J.; Williams, D. P.; Park, B. K.; Stachulski, A. V. Haloarene derivatives of carbamazepine with reduced bioactivation liabilities: 2-monohalo and 2,8-dihalo derivatives. J. Med. Chem. 2012, 55, 9773– 9784, DOI: 10.1021/jm301013n[ACS Full Text], [CAS], Google Scholar319https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFCrur7J&md5=d158092424146266ca119a4d5656ba30Haloarene Derivatives of Carbamazepine with Reduced Bioactivation Liabilities: 2-Monohalo and 2,8-Dihalo DerivativesElliott, Emma-Claire; Regan, Sophie L.; Maggs, James L.; Bowkett, Elizabeth R.; Parry, Laura J.; Williams, Dominic P.; Park, B. Kevin; Stachulski, Andrew V.Journal of Medicinal Chemistry (2012), 55 (22), 9773-9784CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The anticonvulsant carbamazepine (I) is assocd. with adverse drug reactions (ADRs), including hepatotoxicity; oxidative metab. of I has been implicated in the pathogenesis of the ADRs. The authors report the synthesis and evaluation of 2-monohalo and 2,8-dihalo analogs of I that were intended to minimize reactive metabolite formation via arene oxidn. and 10,11-epoxidn. Halo analogs were obtained either by rearrangement of halogenated N-arylindoles or from specifically halogenated iminodibenzyl derivs. In rat hepatocytes, none of the analogs underwent oxidative dehalogenation or glutathione adduction. Some formation of the 10,11-epoxide still occurred, but arom. hydroxylation was not seen with the exception of 2-fluoro, which allowed minor monohydroxylation. Complete inhibition of arom. hydroxylation required at least monochlorination or difluorination of I. In human liver microsomes, difluoro analog II underwent 10,11-epoxidn. but gave no arene oxidn.320Hansch, C.; Leo, A.; Unger, S. H.; Kim, K. H.; Nikaitani, D.; Lien, E. J. ″Aromatic″ substituent constants for structure-activity correlations. J. Med. Chem. 1973, 16, 1207– 1216, DOI: 10.1021/jm00269a003[ACS Full Text], [CAS], Google Scholar320https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3sXlt1Wkt7o%253D&md5=e0ee110086f19b9853838f47ba1c1d4dAromatic substituent constants for structure-activity correlationsHansch, Corwin; Leo, Albert; Unger, Stefan H.; Kim, Ki Hwai; Nikaitani, Donald; Lien, Eric J.Journal of Medicinal Chemistry (1973), 16 (11), 1207-16CODEN: JMCMAR; ISSN:0022-2623.The arom. substituent consts. π (lipophilic), σm and σp (electronic), and molar refractivity (steric) were collected from the literature for 236 substituents, including 128 π values and 191 values for which both σm and σp were found. The field and resonance electronic properties of C. G. Swain and E. C. Lupton (1968) were calcd. for these 191 substituents by a corrected procedure and were essentially orthogonal. The mutual correlation of σm and σp was high.321Samuel, K.; Yin, W.; Stearns, R. A.; Tang, Y. S.; Chaudhary, A. G.; Jewell, J. P.; Lanza, T., Jr.; Lin, L. S.; Hagmann, W. K.; Evans, D. C.; Kumar, S. Addressing the metabolic activation potential of new leads in drug discovery: a case study using ion trap mass spectrometry and tritium labeling techniques. J. Mass Spectrom. 2003, 38, 211– 221, DOI: 10.1002/jms.434[Crossref], [PubMed], [CAS], Google Scholar321https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhsFGmurc%253D&md5=219eb210acef4357bfba39aec0576414Addressing the metabolic activation potential of new leads in drug discovery: A case study using ion trap mass spectrometry and tritium labeling techniquesSamuel, Koppara; Yin, Wenji; Stearns, Ralph A.; Tang, Yui S.; Chaudhary, Ashok G.; Jewell, James P.; Lanza, Thomas, Jr.; Lin, Linus S.; Hagmann, William K.; Evans, David C.; Kumar, SanjeevJournal of Mass Spectrometry (2003), 38 (2), 211-221CODEN: JMSPFJ; ISSN:1076-5174. (John Wiley & Sons Ltd.)Metabolic activation of drug candidates to electrophilic reactive metabolites that can covalently modify cellular macromols. may result in acute and/or idiosyncratic immune system-mediated toxicities in humans. This presents a significant potential liability for the future development of these compds. as safe therapeutic agents. We present here an example of an approach where sites of metabolic activation within a new drug candidate series were rapidly identified using online liq. chromatog./multistage mass spectrometry on an ion trap mass spectrometer. This was accomplished by trapping the reactive intermediates formed upon incubation of compds. with rat and human liver microsomes as their corresponding glutathione conjugates and mass spectral characterization of these thiol adducts. Based on the structures of the GSH adducts identified, potential sites and mechanisms of bioactivation within the chem. structure were proposed. These metab. studies were interfaced with iterative structural modifications of the chem. series in order to block these bioactivation sites within the mol. This strategy led to a significant redn. in the propensity of the compds. to undergo metabolic activation as evidenced by redns. in the irreversible binding of radioactivity to liver microsomal material upon incubation of tritium-labeled compds. with this in vitro system. With the efficiency and throughput achievable with such an approach, it appears feasible to identify and address the metabolic activation potential of new drug leads during routine metabolite identification studies in an early drug discovery setting.322Koerts, J.; Soffers, A. E.; Vervoort, J.; De Jager, A.; Rietjens, I. M. Occurrence of the NIH shift upon the cytochrome P450-catalyzed in vivo and in vitro aromatic ring hydroxylation of fluorobenzenes. Chem. Res. Toxicol. 1998, 11, 503– 512, DOI: 10.1021/tx980053i[ACS Full Text], [CAS], Google Scholar322https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXjt1Giurc%253D&md5=64178da1e0f25584e5aa883b57d3498cOccurrence of the NIH Shift upon the Cytochrome P450-Catalyzed in Vivo and in Vitro Aromatic Ring Hydroxylation of FluorobenzenesKoerts, Janneke; Soffers, Ans E. M. F.; Vervoort, Jacques; De Jager, Adrie; Rietjens, Ivonne M. C. M.Chemical Research in Toxicology (1998), 11 (5), 503-512CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)The in vivo cytochrome P 450-catalyzed arom. hydroxylation of a series of fluorobenzenes was investigated with special emphasis on the importance of the fluorine NIH shift. The results obtained demonstrate a minor role for the NIH shift in the metab. of the fluorobenzenes to phenolic metabolites in control male Wistar rats. These in vivo results could indicate that (1) the NIH shift is an inherently minor process for fluorine substituents or (2) it is a potentially significant process but the presumed epoxide that leads to formation of the NIH-shifted metabolite is lost to an alternative metabolic pathway. In contrast to the in vivo data, in vitro expts. showed a significant amt. of NIH-shifted metabolites for 1,4-difluorobenzene. This result eliminates the explanation that the NIH shift is an inherently minor process for fluorine substituents. Results of addnl. expts. presented in this paper show that the reduced tendency of fluorine-substituted benzenes to undergo an NIH shift in vivo can-at least in part-be ascribed to the possible existence of alternative pathways for metab. of the epoxide, such as, for example, GSH conjugation, being more efficient for fluorinated than chlorinated benzenes.323Dear, G. J.; Ismail, I. M.; Mutch, P. J.; Plumb, R. S.; Davies, L. H.; Sweatman, B. C. Urinary metabolites of a novel quinoxaline non-nucleoside reverse transcriptase inhibitor in rabbit, mouse and human: identification of fluorine NIH shift metabolites using NMR and tandem MS. Xenobiotica 2000, 30, 407– 426, DOI: 10.1080/004982500237604[Crossref], [PubMed], [CAS], Google Scholar323https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXmtFygtbk%253D&md5=ea3b3a235386d9664d5365a5929ed838Urinary metabolites of a novel quinoxaline non-nucleoside reverse transcriptase inhibitor in rabbit, mouse and human: identification of fluorine NIH shift metabolites using NMR and tandem MSDear, G. J.; Ismail, I. M.; Mutch, P. J.; Plumb, R. S.; Davies, L. H.; Sweatman, B. C.Xenobiotica (2000), 30 (4), 407-426CODEN: XENOBH; ISSN:0049-8254. (Taylor & Francis Ltd.)1. The urinary metabolites of (S)-2-ethyl-7-fluoro-3-oxo-3,4-dihydro-2H-quinoxaline-carboxylic acid isopropylester (GW420867X) have been investigated in samples obtained following oral administration to rabbit, mouse and human. GW420867X underwent extensive biotransformation to form hydroxylated metabolites and glucuronide conjugates on the arom. ring, and on the Et and iso-Pr side-chains in all species. In rabbit urine, a minor metabolite was detected and characterized as a cysteine adduct that was not obsd. in mouse or man. 2. The hydroxylated metabolites and corresponding glucuronide conjugates were isolated by semi-preparative HPLC and characterized using NMR, LC-NMR and LC-MS/MS. The relative proportions of fluorine-contg. metabolites were detd. in animal species by 19F-NMR signal integration. 3. The fluorine atom of the arom. ring underwent NIH shift rearrangement in the metabolites isolated and characterized in rabbit, mouse and human urine. 4. The characterization of the NIH shift metabolites in urine enabled the detection and confirmation of the presence of these metabolites in human plasma.324Gunduz, M.; Argikar, U. A.; Kamel, A.; Colizza, K.; Bushee, J. L.; Cirello, A.; Lombardo, F.; Harriman, S. Oxidative ipso substitution of 2,4-difluoro-benzylphthalazines: identification of a rare stable quinone methide and subsequent GSH conjugate. Drug Metab. Dispos. 2012, 40, 2074– 2080, DOI: 10.1124/dmd.112.046268[Crossref], [PubMed], [CAS], Google Scholar324https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFKkt73J&md5=228931ded8e0cae39915b3f0a2f694b0Oxidative ipso substitution of 2,4-difluoro-benzylphthalazines: identification of a rare stable quinone methide and subsequent GSH conjugateGunduz, Mithat; Argikar, Upendra A.; Kamel, Amin; Colizza, Kevin; Bushee, Jennifer L.; Cirello, Amanda; Lombardo, Franco; Harriman, ShawnDrug Metabolism & Disposition (2012), 40 (11), 2074-2080CODEN: DMDSAI; ISSN:1521-009X. (American Society for Pharmacology and Experimental Therapeutics)In vitro metabolite identification and GSH trapping studies in human liver microsomes were conducted to understand the bioactivation potential of compd. 1 [2-(6-(4-(4-(2,4-difluorobenzyl)phthalazin-1-yl)piperazin-1-yl)pyridin-3-yl)propan-2-ol], an inhibitor of the Hedgehog pathway. The results revealed the formation of a unique, stable quinone methide metabolite (M1) via ipso substitution of a fluorine atom and subsequent formation of a GSH adduct (M2). The stability of this metabolite arises from extensive resonance-stabilized conjugation of the substituted benzylphthalazine moiety. Cytochrome P 450 (P 450) phenotyping studies revealed that the formation of M1 and M2 were NADPH-dependent and primarily catalyzed by CYP3A4 among the studied P 450 isoforms. In summary, an unusual and stable quinone methide metabolite of compd. 1 was identified, and a mechanism was proposed for its formation via an oxidative ipso substitution.325Chen, H.; Shockcor, J.; Chen, W.; Espina, R.; Gan, L. S.; Mutlib, A. E. Delineating novel metabolic pathways of DPC 963, a non-nucleoside reverse transcriptase inhibitor, in rats. Characterization of glutathione conjugates of postulated oxirene and benzoquinone imine intermediates by LC/MS and LC/NMR. Chem. Res. Toxicol. 2002, 15, 388– 399, DOI: 10.1021/tx010153f[ACS Full Text], [CAS], Google Scholar325https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XhtFalu7o%253D&md5=ff34e05f9eb59f8ff07ef2698433d166Delineating novel metabolic pathways of DPC 963, a non-nucleoside reverse transcriptase inhibitor, in rats. Characterization of glutathione conjugates of postulated oxirene and benzoquinone imine intermediates by LC/MS and LC/NMRChen, Hao; Shockcor, John; Chen, Weiqi; Espina, Robert; Gan, Liang-Shang; Mutlib, A. E.Chemical Research in Toxicology (2002), 15 (3), 388-399CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)The metabolic activation of (S)-5,6-difluoro-4-cyclopropylethynyl-4-trifluoromethyl-3,4-dihydro-2(1H)-quinazolinone, DPC 963, in rats was investigated by identifying and characterizing the GSH and mercapturic acid conjugates excreted in the bile and urine, resp. The structures of these adducts, which were unequivocally elucidated by LC/MS/MS and NMR expts., revealed the existence of at least three distinct metabolic pathways leading to these products. One of the pathways, which has been described previously, involves the activation of the acetylene group after an initial hydroxylation on the methine carbon of the cyclopropyl ring. Metabolite M1 was demonstrated to be formed via this pathway after an enzymic addn. of GSH across the triple bond of the substituted acetylene. The second pathway, also previously described, leads to diastereoisomeric GSH adducts M3 and M4 after the formation of a highly reactive oxirene intermediate. This postulated oxirene subsequently rearranges to an α, β-unsatd. cyclobutenyl ketone intermediate capable of undergoing a 1,4-Michael addn. with a nucleophile such as GSH. In addn. to these pathways, DPC 963 was found to undergo a metabolic activation previously undescribed for structural analogs of this compd. It is postulated that an oxidative defluorination mediated by cytochrome P 450 leads to the formation of a putative benzoquinone imine intermediate which subsequently reacts with GSH to form two arom. ring-substituted regioisomeric conjugates, M5 and M6. In addn. to forming the GSH adducts, the benzoquinone imine was also found to be reduced to its unreactive hydroquinone metabolite, which was excreted as the glucuronide conjugate in rat bile. Studies with induced rat microsomes, cDNA-expressed rat P 450 isoenzymes, and polyclonal antibodies against rat P 450 clearly demonstrated that the rat P450s 3A1/3A2 were responsible for the formation of postulated oxirene and benzoquinone intermediates.326Hebner, C. M.; Han, B.; Brendza, K. M.; Nash, M.; Sulfab, M.; Tian, Y.; Hung, M.; Fung, W.; Vivian, R. W.; Trenkle, J.; Taylor, J.; Bjornson, K.; Bondy, S.; Liu, X.; Link, J.; Neyts, J.; Sakowicz, R.; Zhong, W.; Tang, H.; Schmitz, U. The HCV non-nucleoside inhibitor Tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase function. PLoS One 2012, 7, e39163 DOI: 10.1371/journal.pone.0039163[Crossref], [PubMed], [CAS], Google Scholar326https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XovF2jtro%253D&md5=55998f37569ff6b657425bf95a0b677dThe HCV non-nucleoside inhibitor tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase functionHebner, Christy M.; Han, Bin; Brendza, Katherine M.; Nash, Michelle; Sulfab, Maisoun; Tian, Yang; Hung, Magdeleine; Fung, Wanchi; Vivian, Randall W.; Trenkle, James; Taylor, James; Bjornson, Kyla; Bondy, Steven; Liu, Xiaohong; Link, John; Neyts, Johan; Sakowicz, Roman; Zhong, Weidong; Tang, Hengli; Schmitz, UliPLoS One (2012), 7 (6), e39163CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Tegobuvir (TGV) is a novel non-nucleoside inhibitor (NNI) of HCV RNA replication with demonstrated antiviral activity in patients with genotype 1 chronic HCV infection. The mechanism of action of TGV has not been clearly defined despite the identification of resistance mutations mapping to the NS5B polymerase region. TGV does not inhibit NS5B enzymic activity in biochem. assays in vitro, suggesting a more complex antiviral mechanism with cellular components. Here, we demonstrate that TGV exerts anti-HCV activity utilizing a unique chem. activation and subsequent direct interaction with the NS5B protein. Treatment of HCV subgenomic replicon cells with TGV results in a modified form of NS5B with a distinctly altered mobility on a SDS-PAGE gel. Further anal. reveals that the aberrantly migrating NS5B species contains the inhibitor mol. Formation of this complex does not require the presence of any other HCV proteins. The intensity of the aberrantly migrating NS5B species is strongly dependent on cellular glutathione levels as well as CYP 1A activity. Furthermore anal. of NS5B protein purified from a heterologous expression system treated with TGV by mass spectrometry suggests that TGV undergoes a CYP-mediated intracellular activation step and the resulting metabolite, after forming a glutathione conjugate, directly and specifically interacts with NS5B. Taken together, these data demonstrate that upon metabolic activation TGV is a specific, covalent inhibitor of the HCV NS5B polymerase and is mechanistically distinct from other classes of the non-nucleoside inhibitors (NNI) of the viral polymerase.327Powers, J. P.; Piper, D. E.; Li, Y.; Mayorga, V.; Anzola, J.; Chen, J. M.; Jaen, J. C.; Lee, G.; Liu, J.; Peterson, M. G.; Tonn, G. R.; Ye, Q.; Walker, N. P.; Wang, Z. SAR and mode of action of novel non-nucleoside inhibitors of hepatitis C NS5b RNA polymerase. J. Med. Chem. 2006, 49, 1034– 1046, DOI: 10.1021/jm050859x[ACS Full Text], [CAS], Google Scholar327https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XksV2ltA%253D%253D&md5=722ac0edb5dabb720e30475ef5925d5fSAR and Mode of Action of Novel Non-Nucleoside Inhibitors of Hepatitis C NS5b RNA PolymerasePowers, Jay P.; Piper, Derek E.; Li, Yang; Mayorga, Veronica; Anzola, John; Chen, James M.; Jaen, Juan C.; Lee, Gary; Liu, Jinqian; Peterson, M. Greg; Tonn, George R.; Ye, Qiuping; Walker, Nigel P. C.; Wang, ZhulunJournal of Medicinal Chemistry (2006), 49 (3), 1034-1046CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Novel non-nucleoside inhibitors of the HCV RNA polymerase (NS5b) with sub-micromolar biochem. potency have been identified which are selective for the inhibition of HCV NS5b over other polymerases. The structures of the complexes formed between several of these inhibitors and HCV NS5b were detd. by x-ray crystallog., and the inhibitors were found to bind in an allosteric binding site sep. from the active site. Structure-activity relationships and structural studies have identified the mechanism of action for compds. in this series, several of which possess drug-like properties, as unique, reversible, covalent inhibitors of HCV NS5b.328Mandal, M.; Mitra, K.; Grotz, D.; Lin, X.; Palamanda, J.; Kumari, P.; Buevich, A.; Caldwell, J. P.; Chen, X.; Cox, K.; Favreau, L.; Hyde, L.; Kennedy, M. E.; Kuvelkar, R.; Liu, X.; Mazzola, R. D.; Parker, E.; Rindgen, D.; Sherer, E.; Wang, H.; Zhu, Z.; Stamford, A. W.; Cumming, J. N. Overcoming time-dependent inhibition (TDI) of cytochrome P450 3A4 (CYP3A4) resulting from bioactivation of a fluoropyrimidine moiety. J. Med. Chem. 2018, 61, 10700– 10708, DOI: 10.1021/acs.jmedchem.8b01326[ACS Full Text], [CAS], Google Scholar328https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitV2rtbfM&md5=09c862a66c3b078e0886696a6c2cd489Overcoming Time-Dependent Inhibition (TDI) of Cytochrome P450 3A4 (CYP3A4) Resulting from Bioactivation of a Fluoropyrimidine MoietyMandal, Mihirbaran; Mitra, Kaushik; Grotz, Diane; Lin, Xinjie; Palamanda, Jairam; Kumari, Pramila; Buevich, Alexei; Caldwell, John P.; Chen, Xia; Cox, Kathleen; Favreau, Leonard; Hyde, Lynn; Kennedy, Matthew E.; Kuvelkar, Reshma; Liu, Xiaoxiang; Mazzola, Robert D.; Parker, Eric; Rindgen, Diane; Sherer, Edward; Wang, Hongwu; Zhu, Zhaoning; Stamford, Andrew W.; Cumming, Jared N.Journal of Medicinal Chemistry (2018), 61 (23), 10700-10708CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Herein the authors describe structure-activity relationship (SAR) and metabolite identification (Met-ID) studies that provided insight into the origin of time-dependent inhibition (TDI) of cytochrome P 450 3A4 (CYP3A4) by compd. I. Collectively, these efforts revealed that bioactivation of the fluoropyrimidine moiety of I led to reactive metabolite formation via oxidative defluorination and was responsible for the obsd. TDI. The authors discovered that substitution at both the 4- and 6-positions of the 5-fluoropyrimidine of I was necessary to ameliorate this TDI as exemplified by compd. II.329Wermers, R. A.; Cooper, K.; Razonable, R. R.; Deziel, P. J.; Whitford, G. M.; Kremers, W. K.; Moyer, T. P. Fluoride excess and periostitis in transplant patients receiving long-term voriconazole therapy. Clin. Infect. Dis. 2011, 52, 604– 611, DOI: 10.1093/cid/ciq188[Crossref], [PubMed], [CAS], Google Scholar329https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhslWmur8%253D&md5=d9af38baae224a2a97d87a8d6693a10dFluoride Excess and Periostitis in Transplant Patients Receiving Long-Term Voriconazole TherapyWermers, Robert A.; Cooper, Kay; Razonable, Raymund R.; Deziel, Paul J.; Whitford, Gary M.; Kremers, Walter K.; Moyer, Thomas P.Clinical Infectious Diseases (2011), 52 (5), 604-611CODEN: CIDIEL; ISSN:1058-4838. (Oxford University Press)We have identified that in transplant subjects, long term use of voriconazole, a fluoride contg. medication, is assocd. with elevated plasma fluoride levels compared to those not taking it. Fluoride related bone complications were also obsd. including periostitis and fluorosis. We describe a heart transplant patient with painful periostitis and exostoses who was receiving long-term therapy with voriconazole, which is a fluoride-contg. medication. Elevated plasma and bone fluoride levels were identified. Discontinuation of voriconazole therapy led to improvement in pain and reduced fluoride and alk. phosphatase levels. To det. whether voriconazole is a cause of fluoride excess, we measured plasma fluoride levels in 10 adult post-transplant patients who had received voriconazole for at least 6 mo and 10 post-transplant patients who did not receive voriconazole. To assess the effect of renal insufficiency on fluoride levels in subjects receiving voriconazole, half were recruited on the basis of a serum creatinine level of 1.4 mg/dL on their most recent measurement, whereas the other 5 subjects receiving voriconazole had serum creatinine levels <1.4 mg/dL. All control subjects had serum creatinine levels of 1.4 mg/dL. Patients were excluded from the study if they received a fluorinated pharmaceutical other than voriconazole. All subjects who received voriconazole had elevated plasma fluoride levels, and no subjects in the control group had elevated levels (14.32 μmol/L ± 6.41 vs 2.54 ± 0.67 μmol/L; P<.001). Renal function was not predictive of fluoride levels. Plasma fluoride levels remained significantly higher in the voriconazole group after adjusting for calcineurin inhibitor levels and doses. Half of the voriconazole group subjects had evidence of periostitis, including exostoses in 2 patients. Discontinuation of voriconazole therapy in patients with periostitis resulted in improvement of pain and a redn. in alk. phosphatase and fluoride levels. Voriconazole is assocd. with painful periostitis, exostoses, and fluoride excess in post-transplant patients with long-term voriconazole use.330Wang, T. F.; Wang, T.; Altman, R.; Eshaghian, P.; Lynch, J. P., 3rd; Ross, D. J.; Belperio, J. A.; Weigt, S. S.; Saggar, R.; Gregson, A.; Kubak, B.; Saggar, R. Periostitis secondary to prolonged voriconazole therapy in lung transplant recipients. Am. J. Transplant. 2009, 9, 2845– 2850, DOI: 10.1111/j.1600-6143.2009.02837.x[Crossref], [PubMed], [CAS], Google Scholar330https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1MfjtlCitw%253D%253D&md5=df2416a750f142fcda08d0a6c460b5c7Periostitis secondary to prolonged voriconazole therapy in lung transplant recipientsWang T F; Wang T; Altman R; Eshaghian P; Lynch J P 3rd; Ross D J; Belperio J A; Weigt S S; Saggar R; Gregson A; Kubak B; Saggar RAmerican journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons (2009), 9 (12), 2845-50 ISSN:.We report five cases of possible drug-induced periostitis associated with long-term use of voriconazole therapy after lung transplantation (LT). The diagnosis of periostitis was made by the documentation of bone pain, elevation of serum alkaline phosphatase and characteristic findings on radionuclide bone imaging in the absence of any identifiable rheumatologic disease. This periostitis appears similar to hypertrophic osteoarthopathy (HOA) but does not meet all criteria for HOA. In all patients, the symptoms resolved rapidly after discontinuation of voriconazole therapy. Awareness of this potential syndrome, which manifests as bone pain, elevated serum alkaline phosphatase and a bone scan suggestive of periostitis, is necessary in LT recipients on long-term voriconazole.331Chen, L.; Mulligan, M. E. Medication-induced periostitis in lung transplant patients: periostitis deformans revisited. Skeletal Radiol 2011, 40, 143– 148, DOI: 10.1007/s00256-010-0997-y[Crossref], [PubMed], [CAS], Google Scholar331https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3M%252Fmt1OnsQ%253D%253D&md5=d44b813266dcdbeb57fe550c341ab117Medication-induced periostitis in lung transplant patients: periostitis deformans revisitedChen Lina; Mulligan Michael ESkeletal radiology (2011), 40 (2), 143-8 ISSN:.We report five cases of diffuse periostitis resembling hypertrophic osteoarthropathy and perostitis deformans in lung transplantation patients on chronic voriconazole, a fluoride-containing compound. Although drug-related periostitis has long been known, the association of lung transplant medication with periostitis was only recently introduced in the literature. To our knowledge, imaging findings have not been fully characterized in the radiology literature. Imaging features along with clinical history help to distinguish this benign condition from other disease entities. In this article, we review the current literature and illustrate the variety of imaging characteristics of this entity so that interpreting radiologists can make accurate diagnoses and avoid unnecessary work up.332Roffey, S. J.; Cole, S.; Comby, P.; Gibson, D.; Jezequel, S. G.; Nedderman, A. N.; Smith, D. A.; Walker, D. K.; Wood, N. The disposition of voriconazole in mouse, rat, rabbit, guinea pig, dog, and human. Drug Metab. Dispos. 2003, 31, 731– 741, DOI: 10.1124/dmd.31.6.731[Crossref], [PubMed], [CAS], Google Scholar332https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXkt1KrtLw%253D&md5=831768b6c72bce3a8528af680c60a3d9The disposition of voriconazole in mouse, rat, rabbit, guinea pig, dog, and humanRoffey, S. J.; Cole, S.; Comby, P.; Gibson, D.; Jezequel, S. G.; Nedderman, A. N. R.; Smith, D. A.; Walker, D. K.; Wood, N.Drug Metabolism and Disposition (2003), 31 (6), 731-741CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)Voriconazole is a new triazole antifungal agent with potent, wide-spectrum activity. Its pharmacokinetics and metab. have been studied in mouse, rat, rabbit, dog, guinea pig, and humans after single and multiple administration by both oral and i.v. routes. Absorption of voriconazole is essentially complete in all species. The elimination of voriconazole is characterized by non-linear pharmacokinetics in all species. Consequently, pharmacokinetic parameters are dependent upon dose, and a superproportional increase in area under the curve is seen with increasing dose in rat and dog toxicol. studies. Following multiple administration, there is a decrease in systemic exposure. This is most pronounced in mouse and rat, less so in dog, and not obsd. in guinea pig or rabbit. Repeat-dose toxicol. studies in mouse, rat, and dog have demonstrated that induction of cytochrome P 450 by voriconazole (autoinduction of metab.) is responsible for the decreased exposure in these species. Autoinduction of metab. is not obsd. in humans, and plasma steady-state concns. remain const. with time. Voriconazole is extensively metabolized in all species. The major pathways in humans involve fluoropyrimidine N-oxidn., fluoropyrimidine hydroxylation, and Me hydroxylation. Also, N-oxidn. facilitates cleavage of the mol., resulting in loss of the fluoropyrimidine moiety and subsequent conjugation with glucuronic acid. Major pathways are represented in animal species. The major circulating metabolite in rat, dog, and human is the N-oxide of voriconazole. It is not thought to contribute to efficacy since it is at least 100-fold less potent than voriconazole against fungal pathogens in vitro.333Schulz, J.; Kluwe, F.; Mikus, G.; Michelet, R.; Kloft, C. Novel insights into the complex pharmacokinetics of voriconazole: a review of its metabolism. Drug Metab. Rev. 2019, 51, 247– 265, DOI: 10.1080/03602532.2019.1632888[Crossref], [PubMed], [CAS], Google Scholar333https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsF2lsrvP&md5=b6209461b8176d373d14b3f90205f69eNovel insights into the complex pharmacokinetics of voriconazole: a review of its metabolismSchulz, Josefine; Kluwe, Franziska; Mikus, Gerd; Michelet, Robin; Kloft, CharlotteDrug Metabolism Reviews (2019), 51 (3), 247-265CODEN: DMTRAR; ISSN:0360-2532. (Taylor & Francis Ltd.)A review. Voriconazole, a second-generation triazole frequently used for the prophylaxis and treatment of invasive fungal infections, undergoes complex metab. mainly involving various (polymorphic) cytochrome P 450 enzymes in humans. Although high inter- and intraindividual variability in voriconazole pharmacokinetics have been obsd. and the therapeutic range for this compd. is relatively narrow, the metab. of voriconazole has not been fully elucidated yet. The available literature data investigating the multiple different pathways and metabolites areextremely unbalanced and thus the abs. or relative contribution of the different pathwaysand enzymes involved in the metab. of voriconazole remains uncertain. Furthermore, other factors such as nonlinear pharmacokinetics caused by auto-inhibition or -induction and polymorphisms of the metabolizing enzymes hinder safe and effective voriconazole dosing in clin. practice and have not yet been studied sufficiently. This review aimed at amalgamating the available literature on the pharmacokinetics of voriconazole in vitro and in vivo, with a special focuson metab. in adults and children, in order to congregate an overall landscape of the currentbody of knowledge and identify knowledge gaps, opening the way towards further research inorder to foster the understanding, towards better therapeutic dosing decisions.334Shang, J.; Xu, S.; Teffera, Y.; Doss, G. A.; Stearns, R. A.; Edmonson, S.; Beconi, M. G. Metabolic activation of a pentafluorophenylethylamine derivative: formation of glutathione conjugates in vitro in the rat. Xenobiotica 2005, 35, 697– 713, DOI: 10.1080/00498250500230479[Crossref], [PubMed], [CAS], Google Scholar334https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1Sns73K&md5=17f4ddc8b005c5ad153497adbba25524Metabolic activation of a pentafluorophenylethylamine derivative: Formation of glutathione conjugates in vitro in the ratShang, J.; Xu, S.; Teffera, Y.; Doss, G. A.; Stearns, R. A.; Edmonson, S.; Beconi, M. G.Xenobiotica (2005), 35 (7), 697-713CODEN: XENOBH; ISSN:0049-8254. (Taylor & Francis Ltd.)The aim was to investigate the metabolic activation potential of a pentafluorophenylethylamine deriv. (compd. I) in vitro in the rat and to identify the cytochrome P 450 (CYP) enzymes that catalyze these metabolic activation processes. Reduced glutathione (GSH) was fortified in rat hepatocytes and liver microsomes to trap possible reactive intermediates. Four glutathione conjugates (M1-4) were identified by LC-MSn following incubation of compd. I in GSH-enriched rat hepatocytes and liver microsomes. Three of these conjugates (M2-4) have not been reported previously for pentafluorophenyl derivs. Elemental compn. anal. of these conjugates was obtained using high-resoln. quadrupole time-of-flight mass spectrometry. The formation of GSH conjugate M1 was rationalized as a direct nucleophilic replacement of fluoride by glutathione, whereas the formation of the GSH conjugates M2-4 was proposed to occur by NADPH-dependent metabolic activation of the pentafluorophenyl ring via arene oxide, quinone and/or quinoneimine reactive intermediates. Formation of these conjugates was enhanced three- to five-fold in liver microsomes obtained from phenobarbital- and dexamethasone-treated rats. In incubations with pooled rat liver microsomes and recombinant rat CYP3A1 and CYP3A2, troleandomycin (TAO) reduced the formation of GSH conjugates M2-4 by 80 - 90%, but it had no effect on the formation of M1. Incubation of compd. I with rat supersomes indicated that only CYP3A1 and CYP3A2 were capable of mediating these metabolic activation processes.335Shan, B.; Medina, J. C.; Santha, E.; Frankmoelle, W. P.; Chou, T. C.; Learned, R. M.; Narbut, M. R.; Stott, D.; Wu, P.; Jaen, J. C.; Rosen, T.; Timmermans, P. B.; Beckmann, H. Selective, covalent modification of β-tubulin residue Cys-239 by T138067, an antitumor agent with in vivo efficacy against multidrug-resistant tumors. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 5686– 5691, DOI: 10.1073/pnas.96.10.5686[Crossref], [PubMed], [CAS], Google Scholar335https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXjtFCntLY%253D&md5=3dd9933e4db4cbb8875e7213c8e86ad8Selective, covalent modification of β-tubulin residue Cys-239 by T138067, an antitumor agent with in vivo efficacy against multidrug-resistant tumorsShan, Bei; Medina, Julio C.; Santha, Edit; Frankmoelle, Walter P.; Chou, Ting-C.; Learned, Robert M.; Narbut, Mathew R.; Stott, Dean; Wu, Pengguang; Jaen, Juan C.; Rosen, Terry; Timmermans, Pieter B. M. W. M.; Beckmann, HolgerProceedings of the National Academy of Sciences of the United States of America (1999), 96 (10), 5686-5691CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Microtubules are linear polymers of α- and β-tubulin heterodimers and are the major constituents of mitotic spindles, which are essential for the sepn. of chromosomes during mitosis. Here we describe a synthetic compd., 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene (T138067), which covalently and selectively modifies the β1, β2, and β4 isotypes of β-tubulin at a conserved cysteine residue, thereby disrupting microtubule polymn. Cells exposed to T138067 become altered in shape, indicating a collapse of the cytoskeleton, and show an increase in chromosomal ploidy. Subsequently, these cells undergo apoptosis. Furthermore, T138067 exhibits cytotoxicity against tumor cell lines that exhibit substantial resistance to vinblastine, paclitaxel, doxorubicin, and actinomycin D. T138067 is also equally efficacious in inhibiting the growth of sensitive and multidrug-resistant human tumor xenografts in athymic nude mice. These observations suggest that T138067 may be clin. useful for the treatment of multidrug-resistant tumors.336Frankmoelle, W. P.; Medina, J. C.; Shan, B.; Narbut, M. R.; Beckmann, H. Glutathione S-transferase metabolism of the antineoplastic pentafluorophenylsulfonamide in tissue culture and mice. Drug Metab. Dispos. 2000, 28, 951– 958[PubMed], [CAS], Google Scholar336https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXlt1aht7w%253D&md5=8045e24a133a8c1d754df9c9d45cb665Glutathione S-transferase metabolism of the antineoplastic pentafluorophenylsulfonamide in tissue culture and miceFrankmoelle, Walter P.; Medina, Julio C.; Shan, Bei; Narbut, Mathew R.; Beckmann, HolgerDrug Metabolism and Disposition (2000), 28 (8), 951-958CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)The microtubule disrupting agent 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene (T138067) binds covalently and selectively to β-tubulin and has been shown to evade drug-efflux pumps that confer multidrug resistance to other antimitotic drugs that are used in cancer chemotherapy. In addn. to these resistance mechanisms, eukaryotic cells have developed other protection mechanisms that involve enzymes that modify electrophilic xenobiotics. To det. whether T138067 is a substrate for such enzymic detoxification pathways, a metab. study was initiated. GSH conjugation was shown to play a major role in T138067 metab. T138067-GSH conjugates were isolated from the culture media of T138067-treated cells and the bile of mice treated i.v. with T138067. The major T138067-GSH degrdn. products were also isolated from these sources. 19F NMR studies of the metabolites showed that metabolic conversions occurred through substitution of the para fluorine atom in the pentafluorophenyl ring of T138067. The T138067-GSH conjugate was also isolated from T138067 incubation buffer that had been exposed to mouse, rat, dog, or human liver slices, suggesting that this mechanism is not species-specific. All three human glutathione S-transferases (α, μ, and π), which are expressed in a wide variety of tissues including human tumors, were shown to metabolize T138067 effectively in vitro. The combined data show that T138067 is being metabolized, in vitro and in vivo, predominantly via a glutathione S-transferase-mediated metabolic pathway.337Yang, J.; Li, Y.; Yan, W.; Li, W.; Qiu, Q.; Ye, H.; Chen, L. Covalent modification of Cys-239 in β-tubulin by small molecules as a strategy to promote tubulin heterodimer degradation. J. Biol. Chem. 2019, 294, 8161– 8170, DOI: 10.1074/jbc.RA118.006325[Crossref], [PubMed], [CAS], Google Scholar337https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtFOnurfP&md5=e92427e7fe735c00f366af467eafe233Covalent modification of Cys-239 in β-tubulin by small molecules as a strategy to promote tubulin heterodimer degradationYang, Jianhong; Li, Yong; Yan, Wei; Li, Weimin; Qiu, Qiang; Ye, Haoyu; Chen, LijuanJournal of Biological Chemistry (2019), 294 (20), 8161-8170CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Clin. microtubule-targeting drugs are functionally divided into microtubule-destabilizing and microtubule-stabilizing agents. Drugs from both classes achieve microtubule inhibition by binding different sites on tubulin and inhibiting or promoting polymn. with no concomitant effects on the protein levels of tubulin heterodimers. Here, we have identified a series of small mols. with diverse structures potentially representing a third class of novel tubulin inhibitors that promote degrdn. by covalent binding to Cys-239 of β-tubulin. The small mols. highlighted in this study include T0070907 (a peroxisome proliferator-activated receptor γ inhibitor), T007-1 (a T0070907 deriv.), T138067, N,N'-ethylene-bis(iodoacetamide) (EBI), and allyl isothiocyanate (AITC). Label-free quant. proteomic anal. revealed that T007-1 promotes tubulin degrdn. with high selectivity. Mass spectrometry findings showed covalent binding of both T0070907 and T007-01 to Cys-239 of β-tubulin. Furthermore, T007-1 exerted a degradative effect on tubulin isoforms possessing Cys-239 (β2, β4, and β5(β)) but not those contg. Ser-239 (β3, β6) or mutant β-tubulin with a C239S substitution. Three small mols. (T138067, EBI, and AITC) also reported to bind covalently to Cys-239 of β-tubulin similarly induced tubulin degrdn. Our results strongly suggest that covalent modification of Cys-239 of β-tubulin by small mols. could serve as a novel strategy to promote tubulin heterodimer degrdn. We propose that these small mols. represent a third novel class of tubulin inhibitor agents that exert their effects through degrdn. activity.338Peterson, E. A.; Teffera, Y.; Albrecht, B. K.; Bauer, D.; Bellon, S. F.; Boezio, A.; Boezio, C.; Broome, M. A.; Choquette, D.; Copeland, K. W.; Dussault, I.; Lewis, R.; Lin, M. H.; Lohman, J.; Liu, J.; Potashman, M.; Rex, K.; Shimanovich, R.; Whittington, D. A.; Vaida, K. R.; Harmange, J. C. Discovery of potent and selective 8-fluorotriazolopyridine c-Met inhibitors. J. Med. Chem. 2015, 58, 2417– 2430, DOI: 10.1021/jm501913a[ACS Full Text], [CAS], Google Scholar338https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtV2nt70%253D&md5=ae86b1c6fac93caa1deecd89410bd837Discovery of Potent and Selective 8-Fluorotriazolopyridine c-Met InhibitorsPeterson, Emily A.; Teffera, Yohannes; Albrecht, Brian K.; Bauer, David; Bellon, Steven F.; Boezio, Alessandro; Boezio, Christiane; Broome, Martin A.; Choquette, Deborah; Copeland, Katrina W.; Dussault, Isabelle; Lewis, Richard; Lin, Min-Hwa Jasmine; Lohman, Julia; Liu, Jingzhou; Potashman, Michele; Rex, Karen; Shimanovich, Roman; Whittington, Douglas A.; Vaida, Karina R.; Harmange, Jean-ChristopheJournal of Medicinal Chemistry (2015), 58 (5), 2417-2430CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The overexpression of c-Met and/or hepatocyte growth factor (HGF), the amplification of the MET gene, and mutations in the c-Met kinase domain can activate signaling pathways that contribute to cancer progression by enabling tumor cell proliferation, survival, invasion, and metastasis. Herein, the authors report the discovery of 8-fluorotriazolopyridines as inhibitors of c-Met activity. Optimization of the 8-fluorotriazolopyridine scaffold through the combination of structure-based drug design, SAR studies, and metabolite identification provided potent (cellular IC50 < 10 nM) selective inhibitors of c-Met with desirable pharmacokinetic properties, e.g. I, that demonstrate potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.339Prasad, V.; Birzin, E. T.; McVaugh, C. T.; Van Rijn, R. D.; Rohrer, S. P.; Chicchi, G.; Underwood, D. J.; Thornton, E. R.; Smith, A. B., 3rd; Hirschmann, R. Effects of heterocyclic aromatic substituents on binding affinities at two distinct sites of somatostatin receptors. Correlation with the electrostatic potential of the substituents. J. Med. Chem. 2003, 46, 1858– 1869, DOI: 10.1021/jm0205088[ACS Full Text], [CAS], Google Scholar339https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXivFagt70%253D&md5=db7d7f8255fae153a3cfd9912dceb1f4Effects of Heterocyclic Aromatic Substituents on Binding Affinities at Two Distinct Sites of Somatostatin Receptors. Correlation with the Electrostatic Potential of the SubstituentsPrasad, Vidya; Birzin, Elizabeth T.; McVaugh, Cheryl T.; van Rijn, Rachel D.; Rohrer, Susan P.; Chicchi, Gary; Underwood, Dennis J.; Thornton, Edward R.; Smith, Amos B., III; Hirschmann, RalphJournal of Medicinal Chemistry (2003), 46 (10), 1858-1869CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)In our continuing program exploring glucose-based peptidomimetics of somatostatin (SRIF-14), we sought to improve the water soly. of our glycosides. This led to insights into the nature of the ligand binding sites at the SRIF receptor. Replacement of the C4 benzyl substituent in glucoside (+)-2 with pyridinylmethyl or pyrazin-2-ylmethyl congeners increased water soly. and enhanced affinity for the human SRIF subtype receptor 4 (sst4). We attribute this effect to hydrogen bond formation. The pyridin-3-ylmethyl substituent at C4, when combined with the imidazol-4-ylmethyl group at C2, generated (-)-19, which has the highest affinity of a glucose-based peptidomimetic at a human SRIF receptor to date (Ki 53 ± 23 nM, n = 6 at sst4). The C4 heterocyclic congeners of glucosides bearing a 1-methoxy substituent rather than an indole side chain at the anomeric carbon, such as (+)-16, also provided information about the Trp8 binding pocket. We correlated the SARs at both the C4 and the Trp8 binding pockets with calcns. of the electrostatic potentials of the diverse C4 arom. substituents using Spartan 3-21G(*) MO anal. These calcns. provide an approx. anal. of a mol.'s ability to interact within a receptor binding site. Our binding studies show that benzene and indole rings, but not pyridinylmethyl nor pyrazin-2-ylmethyl rings, can bind the hydrophobic Trp8 binding pocket of sst4. The Spartan 3-21G(*) MO anal. reveals significant neg. electrostatic potential in the region of the π-clouds for the benzene and indole rings but not for the pyridinylmethyl or pyrazin-2-ylmethyl congeners. Our data further demonstrate that the replacement of benzene or indole side chains by heterocyclic arom. rings typified by pyridine and pyrazine not only enhances water soly. and hydrogen bonding capacity as expected, but can also profoundly diminish the ability of the π-cloud of the arom. substituent to interact with side chains of an arom. binding pocket such as that for Trp8 of SRIF-14. Conversely, these calcns. accommodate the exptl. findings that pyrazin-2-ylmethyl and pyridinylmethyl substituents at C4- of C1-indole-substituted glycosides afford higher affinities at sst4 than the C4-benzyl group of (+)-2. This result is consistent with the high electron d. in the plane of the heterocycle depicted in Figure 6 which can accept hydrogen bonds from the C4 binding pocket of the receptor. Unexpectedly, we found that the 2-fluoropyridin-5-ylmethyl analog (+)-14 more closely resembles the binding affinity of (+)-8 than that of (+)-2, thus suggesting that (+)-14 represents a rare example of a carbon linked fluorine atom acting as a hydrogen bond acceptor. We attribute this result to the ability of the proton to bind the nitrogen and fluorine atoms simultaneously in a bifurcated arrangement. At the NK1 receptor of substance P (SP), the free hydroxyl at C4 optimizes affinity.340Leach, A. G.; Kidley, N. J. Quantitatively interpreted enhanced inhibition of cytochrome P450s by heteroaromatic rings containing nitrogen. J. Chem. Inf. Model. 2011, 51, 1048– 1063, DOI: 10.1021/ci2000506[ACS Full Text], [CAS], Google Scholar340https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXlsFyktbc%253D&md5=9b37fed557509c4c40c4521797bea92eQuantitatively Interpreted Enhanced Inhibition of Cytochrome P450s by Heteroaromatic Rings Containing NitrogenLeach, Andrew G.; Kidley, Nathan J.Journal of Chemical Information and Modeling (2011), 51 (5), 1048-1063CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)It has been known for a long time that certain substructures bind to the heme iron in cytochromes P 450. Detection of spectroscopic changes and crystal structures of protein ligand complexes have provided qual. evidence, including for arom. nitrogen-contg. ligands. Compds. contg. these same substructures are more likely to inhibit cytochrome P450s than expected due to lipophilicity. These two sets of observations are not easily linked by expt., because binding to the iron atom alone is not readily measured. Quantum mech. (d. functional) calcns. of binding energies for a no. of different arom. heterocycles to heme iron in a range of oxidn. and spin states can provide a quant. link between the obsd. structures and the biochem. inhibition that is measured. The studies reported here for a set of heteroarom. rings contg. nitrogen begin with quantum mech. calcns. which provide geometries and binding energies. Subsequently, AstraZeneca's database of cytochrome P 450 inhibition assays has been searched to find data that are relevant to the same set of heteroarom. compds. These data have been analyzed in a no. of fashions to account for both the narrow dynamic range of the assays and the lipophilicity dependence of this kind of inhibition. Finally, crystal structures have provided exptl. geometric information. Taken together these different sources suggest that binding to the metal in our inhibition assays is dominated by FeIII in its doublet state, most likely occurring when the iron is pentavalent. Computed binding energies to this state contrast with the hydrogen-bond acceptor ability and basicity of the compds., neither of which are able to correctly account for the effect of the particular environment in which the iron is found. This highlights the value of modeling biochem. events as closely as can be computationally afforded. The computational protocol devised was used to make predictions about a set of as yet unknown heteroarom. compds. suggested by Pitt et al.341Green, J.; Cao, J.; Bandarage, U. K.; Gao, H.; Court, J.; Marhefka, C.; Jacobs, M.; Taslimi, P.; Newsome, D.; Nakayama, T.; Shah, S.; Rodems, S. Design, synthesis, and structure-activity relationships of pyridine-based rho kinase (ROCK) inhibitors. J. Med. Chem. 2015, 58, 5028– 5037, DOI: 10.1021/acs.jmedchem.5b00424[ACS Full Text], [CAS], Google Scholar341https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXpsFars7k%253D&md5=7839e593cb7add8c828cbb4ac49ed674Design, Synthesis, and Structure-Activity Relationships of Pyridine-Based Rho Kinase (ROCK) InhibitorsGreen, Jeremy; Cao, Jingrong; Bandarage, Upul K.; Gao, Huai; Court, John; Marhefka, Craig; Jacobs, Marc; Taslimi, Paul; Newsome, David; Nakayama, Tomoko; Shah, Sundeep; Rodems, SteveJournal of Medicinal Chemistry (2015), 58 (12), 5028-5037CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The Rho kinases (ROCK1 and ROCK2) are highly homologous serine/threonine kinases that act on substrates assocd. with cellular motility, morphol., and contraction and are of therapeutic interest in diseases assocd. with cellular migration and contraction, such as hypertension, glaucoma, and erectile dysfunction. Beginning with compd. 4, an inhibitor of ROCK1 identified through high-throughput screening, systematic exploration of SAR, and application of structure-based design, led to potent and selective ROCK inhibitors. Compd. 37 represents significant improvements in inhibition potency, kinase selectivity, and CYP inhibition and possesses pharmacokinetics suitable for in vivo experimentation.342Dolle, F. [18F]fluoropyridines: From conventional radiotracers to the labeling of macromolecules such as proteins and oligonucleotides. Ernst Schering Res. Found. Workshop 2007, 64, 113– 157, DOI: 10.1007/978-3-540-49527-7_5343Kumar, J. S.; Walker, M.; Packiarajan, M.; Jubian, V.; Prabhakaran, J.; Chandrasena, G.; Pratap, M.; Parsey, R. V.; Mann, J. J. Radiosynthesis and in vivo evaluation of neuropeptide Y5 receptor (NPY5R) PET tracers. ACS Chem. Neurosci. 2016, 7, 540– 545, DOI: 10.1021/acschemneuro.5b00315[ACS Full Text], [CAS], Google Scholar343https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xis1Citrs%253D&md5=3b6e852390caf4c665b8a327a0045481Radiosynthesis and in Vivo Evaluation of Neuropeptide Y5 Receptor (NPY5R) PET TracersKumar, J. S. Dileep; Walker, Mary; Packiarajan, Mathivanan; Jubian, Vrej; Prabhakaran, Jaya; Chandrasena, Gamini; Pratap, Mali; Parsey, Ramin V.; Mann, J. JohnACS Chemical Neuroscience (2016), 7 (5), 540-545CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)Neuropeptide Y receptor type 5 (NPY5R) is a G-protein coupled receptor (GPCR) that belongs to the subfamily of neuropeptide receptors (NPYR) that mediate the action of endogenous neuropeptide Y (NPY). Animal models and preclin. studies indicate a role for NPY5R in the pathophysiol. of depression, anxiety, and obesity and as a target of potential therapeutic drugs. To better understand the pathophysiol. involvement of NPY5R, and to measure target occupancy by potential therapeutic drugs, it would be advantageous to measure NPY5R binding in vivo by positron emission tomog. (PET). Four potent and selective NPY5R antagonists were radiolabeled via nucleophilic arom. substitution reactions with [18F]fluoride. Of the four radioligands investigated, PET studies in anesthetized baboons showed that [18F]LuAE00654 ([18F]N-[trans-4-({[4-(2-fluoropyridin-3-yl)thiazol-2-yl]amino}methyl)cyclohexyl]propane-2-sulfonamide) penetrates blood brain barrier (BBB) and a small amt. is retained in the brain. Slow metab. of [18F]LuAE00654 was obsd. in baboon plasma. Blocking studies with a specific NPY5R antagonist demonstrated up to 60% displacement of radioactivity in striatum, the brain region with highest NPY5R binding. Our studies suggest that [18F]LuAE00654 can be a potential PET radiotracer for the quantification and occupancy studies of NPY5R drug candidates.344Fujinaga, M.; Luo, R.; Kumata, K.; Zhang, Y.; Hatori, A.; Yamasaki, T.; Xie, L.; Mori, W.; Kurihara, Y.; Ogawa, M.; Nengaki, N.; Wang, F.; Zhang, M. R. Development of a (18)F-labeled radiotracer with improved brain kinetics for positron emission tomography imaging of translocator protein (18 kDa) in ischemic brain and glioma. J. Med. Chem. 2017, 60, 4047– 4061, DOI: 10.1021/acs.jmedchem.7b00374[ACS Full Text], [CAS], Google Scholar344https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmt1amtLk%253D&md5=851513be4dbb048ec16135700e33cf36Development of a 18F-Labeled Radiotracer with Improved Brain Kinetics for Positron Emission Tomography Imaging of Translocator Protein (18 kDa) in Ischemic Brain and GliomaFujinaga, Masayuki; Luo, Rui; Kumata, Katsushi; Zhang, Yiding; Hatori, Akiko; Yamasaki, Tomoteru; Xie, Lin; Mori, Wakana; Kurihara, Yusuke; Ogawa, Masanao; Nengaki, Nobuki; Wang, Feng; Zhang, Ming-RongJournal of Medicinal Chemistry (2017), 60 (9), 4047-4061CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)We designed four novel acetamidobenzoxazolone compds. 7a-d as candidates for positron emission tomog. (PET) radiotracers for imaging the translocator protein (18 kDa, TSPO) in ischemic brain and glioma. Among these compds., 2-(5-(6-fluoropyridin-3-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamide (7d) exhibited high binding affinity (Ki = 13.4 nM) with the TSPO and moderate lipophilicity (log D = 1.92). [18F]7d was radiosynthesized by [18F]fluorination of the bromopyridine precursor 7h with [18F]F- in 12 ± 5% radiochem. yield (n = 6, decay-cor.). In vitro autoradiog. and PET studies of ischemic rat brain revealed higher binding of [18F]7d with TSPO on the ipsilateral side, as compared to the contralateral side, and improved brain kinetics compared with our previously developed radiotracers. Metabolite study of [18F]7d showed 93% of unchanged form in the ischemic brain at 30 min after injection. Moreover, PET study with [18F]7d provided a clear tumor image in a glioma-bearing rat model. We demonstrated that [18F]7d is a useful PET radiotracer for visualizing not only neuroinflammation but also glioma and will translate this radiotracer to a "first-in-human" study in our facility.345Naik, R.; Valentine, H.; Dannals, R. F.; Wong, D. F.; Horti, A. G. Synthesis and evaluation of a new (18)F-labeled radiotracer for studying the GABAB receptor in the mouse brain. ACS Chem. Neurosci. 2018, 9, 1453– 1461, DOI: 10.1021/acschemneuro.8b00038[ACS Full Text], [CAS], Google Scholar345https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjslKrtrc%253D&md5=40518bd59f1589a4346688967f279e2eSynthesis and Evaluation of a New 18F-Labeled Radiotracer for Studying the GABAB Receptor in the Mouse BrainNaik, Ravi; Valentine, Heather; Dannals, Robert F.; Wong, Dean F.; Horti, Andrew G.ACS Chemical Neuroscience (2018), 9 (6), 1453-1461CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)New GABAB agonists, fluoropyridyl ether analogs of baclofen, have been synthesized as potential PET radiotracers. The compd. with highest inhibition binding affinity as well as greatest agonist response, (R)-4-amino-3-(4-chloro-3-((2-fluoropyridin-4-yl)methoxy)phenyl)butanoic acid (1b), was radiolabeled with 18F with good radiochem. yield, high radiochem. purity, and high molar radioactivity. The regional brain distribution of the radiolabeled (R)-4-amino-3-(4-chloro-3-((2-[18F]fluoropyridin-4-yl)methoxy)phenyl)butanoic acid, [18F]1b, was studied in CD-1 male mice. The study demonstrated that [18F]1b enters the mouse brain (1% ID/g tissue). The accumulation of [18F]1b in the mouse brain was inhibited (35%) by preinjection of GABAB agonist 1a, suggesting that the radiotracer brain uptake is partially mediated by GABAB receptors. The presented data demonstrate a feasibility of imaging of GABAB receptors in rodents and justify further development of GABAB PET tracers with improved specific binding and greater blood-brain barrier permeability.346Ward, S. E.; Harries, M.; Aldegheri, L.; Andreotti, D.; Ballantine, S.; Bax, B. D.; Harris, A. J.; Harker, A. J.; Lund, J.; Melarange, R.; Mingardi, A.; Mookherjee, C.; Mosley, J.; Neve, M.; Oliosi, B.; Profeta, R.; Smith, K. J.; Smith, P. W.; Spada, S.; Thewlis, K. M.; Yusaf, S. P. Discovery of N-[(2S)-5-(6-fluoro-3-pyridinyl)-2,3-dihydro-1H-inden-2-yl]-2-propanesulfonamide, a novel clinical AMPA receptor positive modulator. J. Med. 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(American Chemical Society)A series of AMPA receptor pos. allosteric modulators has been optimized from poorly penetrant leads to identify mols. with excellent preclin. pharmacokinetics and CNS penetration. These discoveries led to 17i (I), a potent, efficacious CNS penetrant mol. with an excellent pharmacokinetic profile across preclin. species, which is well tolerated and is also orally bioavailable in humans.347Li, P.; Zheng, H.; Zhao, J.; Zhang, L.; Yao, W.; Zhu, H.; Beard, J. D.; Ida, K.; Lane, W.; Snell, G.; Sogabe, S.; Heyser, C. J.; Snyder, G. L.; Hendrick, J. P.; Vanover, K. E.; Davis, R. E.; Wennogle, L. P. Discovery of potent and selective inhibitors of phosphodiesterase 1 for the treatment of cognitive impairment associated with neurodegenerative and neuropsychiatric diseases. J. Med. Chem. 2016, 59, 1149– 1164, DOI: 10.1021/acs.jmedchem.5b01751[ACS Full Text], [CAS], Google Scholar347https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1aisLk%253D&md5=73741521722dc906869ffa3504ceaf2eDiscovery of Potent and Selective Inhibitors of Phosphodiesterase 1 for the Treatment of Cognitive Impairment Associated with Neurodegenerative and Neuropsychiatric DiseasesLi, Peng; Zheng, Hailin; Zhao, Jun; Zhang, Lei; Yao, Wei; Zhu, Hongwen; Beard, J. David; Ida, Koh; Lane, Weston; Snell, Gyorgy; Sogabe, Satoshi; Heyser, Charles J.; Snyder, Gretchen L.; Hendrick, Joseph P.; Vanover, Kimberly E.; Davis, Robert E.; Wennogle, Lawrence P.Journal of Medicinal Chemistry (2016), 59 (3), 1149-1164CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A diverse set of 3-aminopyrazolo[3,4-d]pyrimidinones was designed and synthesized. The structure-activity relationships of these polycyclic compds. as phosphodiesterase 1 (PDE1) inhibitors were studied along with their physicochem. and pharmacokinetic properties. Systematic optimizations of this novel scaffold culminated in the identification of a clin. candidate I, which exhibited picomolar inhibitory potency for PDE1, demonstrated excellent selectivity against all other PDE families and showed good efficacy in vivo. Currently, this investigational new drug is in Phase I clin. development and being considered for the treatment of several indications including cognitive deficits assocd. with schizophrenia and Alzheimer's disease, movement disorders, attention deficit and hyperactivity disorders, and other central nervous system (CNS) and non-CNS disorders.348Wittman, M. D.; Carboni, J. M.; Yang, Z.; Lee, F. Y.; Antman, M.; Attar, R.; Balimane, P.; Chang, C.; Chen, C.; Discenza, L.; Frennesson, D.; Gottardis, M. M.; Greer, A.; Hurlburt, W.; Johnson, W.; Langley, D. R.; Li, A.; Li, J.; Liu, P.; Mastalerz, H.; Mathur, A.; Menard, K.; Patel, K.; Sack, J.; Sang, X.; Saulnier, M.; Smith, D.; Stefanski, K.; Trainor, G.; Velaparthi, U.; Zhang, G.; Zimmermann, K.; Vyas, D. M. Discovery of a 2,4-disubstituted pyrrolo[1,2-f][1,2,4]triazine inhibitor (BMS-754807) of insulin-like growth factor receptor (IGF-1R) kinase in clinical development. J. Med. Chem. 2009, 52, 7360– 7363, DOI: 10.1021/jm900786r[ACS Full Text], [CAS], Google Scholar348https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtFOiurjJ&md5=be0fe9adea18595b4ba6f232726ba4dfDiscovery of a 2,4-Disubstituted Pyrrolo[1,2-f][1,2,4]triazine Inhibitor (BMS-754807) of Insulin-like Growth Factor Receptor (IGF-1R) Kinase in Clinical DevelopmentWittman, Mark D.; Carboni, Joan M.; Yang, Zheng; Lee, Francis Y.; Antman, Melissa; Attar, Ricardo; Balimane, Praveen; Chang, Chiehying; Chen, Cliff; Discenza, Lorell; Frennesson, David; Gottardis, Marco M.; Greer, Ann; Hurlburt, Warren; Johnson, Walter; Langley, David R.; Li, Aixin; Li, Jianqing; Liu, Peiying; Mastalerz, Harold; Mathur, Arvind; Menard, Krista; Patel, Karishma; Sack, John; Sang, Xiaopeng; Saulnier, Mark; Smith, Daniel; Stefanski, Kevin; Trainor, George; Velaparthi, Upender; Zhang, Guifen; Zimmermann, Kurt; Vyas, Dolatrai M.Journal of Medicinal Chemistry (2009), 52 (23), 7360-7363CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)This report describes the biol. activity, characterization, and SAR leading to 9d (BMS-754807, I) a small mol. IGF-1R kinase inhibitor in clin. development.349Ndubaku, C. O.; Crawford, T. D.; Chen, H.; Boggs, J. W.; Drobnick, J.; Harris, S. F.; Jesudason, R.; McNamara, E.; Nonomiya, J.; Sambrone, A.; Schmidt, S.; Smyczek, T.; Vitorino, P.; Wang, L.; Wu, P.; Yeung, S.; Chen, J.; Chen, K.; Ding, C. Z.; Wang, T.; Xu, Z.; Gould, S. E.; Murray, L. J.; Ye, W. Structure-based design of GNE-495, a potent and selective MAP4K4 inhibitor with efficacy in retinal angiogenesis. ACS Med. Chem. 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(American Chemical Society)Diverse biol. roles for mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) have necessitated the identification of potent inhibitors to study its function in various disease contexts. In particular, compds. that can be used to carry out such studies in vivo would be crit. for elucidating the potential for therapeutic intervention. A structure-based design effort coupled with property-guided optimization directed at minimizing the ability of the inhibitors to cross into the CNS led to an advanced compd. (I; GNE-495) that showed excellent potency and good PK and was used to demonstrate in vivo efficacy in a retinal angiogenesis model recapitulating effects that were obsd. in the inducible Map4k4 knockout mice.350Clark, H. R.; Beth, L. D.; Burton, R. M.; Garrett, D. L.; Miller, A. L.; Muscio, O. J., Jr. Kinetic study of the acid-promoted hydrolysis of some representative 2-fluoro nitrogen heterocycles. J. Org. 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These results were correlated with the decline in water activity by means of the Bunnett w and w* relationships, as well as the Bunnett-Olsen LFER. The slopes of these correlations were suggestive of a proton-transfer role for water in the reactions of the less activated 2-fluoropyridines and of 2-fluoroquinoline, while the correlations indicate a nucleophilic role for water in the reactions of the more highly activated pyrimidines. Entropies of activation were significantly more neg. for the pyridine and quinoline systems than for the pyrimidines. The results are interpreted as consistent with nucleophilic attack by water in the rate-detg. step for the reaction of the pyrimidines, while for the less activated substrates nucleophilic attack may be assisted by proton transfer to addnl. water mols.351Inoue, K.; Ohe, T.; Mori, K.; Sagara, T.; Ishii, Y.; Chiba, M. Aromatic substitution reaction of 2-chloropyridines catalyzed by microsomal glutathione S-transferase 1. Drug Metab. Dispos. 2009, 37, 1797– 1800, DOI: 10.1124/dmd.109.027698[Crossref], [PubMed], [CAS], Google Scholar351https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVOrsrnN&md5=080edfa7114d4b506d3376e514b11ab7Aromatic substitution reaction of 2-chloropyridines catalyzed by microsomal glutathione S-transferase 1Inoue, Kazuko; Ohe, Tomoyuki; Mori, Kenichi; Sagara, Takeshi; Ishii, Yasuyuki; Chiba, MasatoDrug Metabolism and Disposition (2009), 37 (9), 1797-1800CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)We investigated the substitution reaction of a series of 2-chloropyridine derivs. catalyzed by rat liver microsomal glutathione S-transferase 1. Various 2-chloropyridine derivs. were metabolized to the corresponding substituted glutathione conjugates via displacement of chlorine atom with glutathione. The reaction was affected by the electron-withdrawing strength and position of the substituents. MO calcns. on the change in Gibbs free energy between the initial and transition states verified the presence of a Meisenheimer complex and its influence on the reaction rate.352Earl, R. A.; Myers, M. J.; Johnson, A. L.; Scribner, R. M.; Wuonola, M. A.; Boswell, G. A.; Wilkerson, W. W.; Nickolson, V. J.; Tam, S. W.; Brittelli, D. R.; Chorvat, R. J.; Zaczek, R.; Cook, L.; Wang, C.; Zhang, X.; Lan, R.; Mi, B.; Wenting, H. Acetylcholine-releasing agents as cognition enhancers. Structure-activity relationships of pyridinyl pendant groups on selected core structures. Bioorg. Med. Chem. Lett. 1992, 2, 851– 854, DOI: 10.1016/S0960-894X(00)80543-0[Crossref], [CAS], Google Scholar352https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXkvVGns74%253D&md5=1d38c0fff1828d31e48c4f84d19fe783Acetylcholine-releasing agents as cognition enhancers. Structure-activity relationships of pyridinyl pendant groups on selected core structuresEarl, Richard A.; Myers, Melvyn J.; Johnson, Alexander L.; Scribner, Richard M.; Wuonola, Mark A.; Boswell, George A.; Wilkerson, Wendell W.; Nickolson, Victor J.; Tam, S. William; et al.Bioorganic & Medicinal Chemistry Letters (1992), 2 (8), 851-4CODEN: BMCLE8; ISSN:0960-894X.A no. of analogs of the cognition enhancing agent DuP 996 (I) were prepd. by varying the core structure and pendant groups in an independent fashion. The structure-activity relations of 2-, 3-, and 4-pyridinylmethyl groups as pendant groups on selected cores was examd.353Wilkerson, W. W.; Kergaye, A. A.; Tam, S. W. 3-Substituted, 3-(4-pyridinylmethyl)-1,3-dihydro-1-phenyl-2H-indol-2-ones as acetylcholine release enhancers: synthesis and SAR. J. Med. 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An attempt has been made to correlate the structure of the 3-substitution with the ability of the compds. to enhance the release of AcCh from the striatum region of rat brain prepns. Alkylation of I (R = H) or its hydrochloride salt gave several of the compds.354Earl, R. A.; Zaczek, R.; Teleha, C. A.; Fisher, B. N.; Maciag, C. M.; Marynowski, M. E.; Logue, A. R.; Tam, S. W.; Tinker, W. J.; Huang, S. M.; Chorvat, R. J. 2-Fluoro-4-pyridinylmethyl analogues of linopirdine as orally active acetylcholine release-enhancing agents with good efficacy and duration of action. J. Med. Chem. 1998, 41, 4615– 4622, DOI: 10.1021/jm9803424[ACS Full Text], [CAS], Google Scholar354https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXmsVKqu7w%253D&md5=f5af93f21d02feedf99d077b178e76fb2-Fluoro-4-pyridinylmethyl Analogs of Linopirdine as Orally Active Acetylcholine Release-Enhancing Agents with Good Efficacy and Duration of ActionEarl, Richard A.; Zaczek, Robert; Teleha, Christopher A.; Fisher, Barbara N.; Maciag, Carla M.; Marynowski, Maria E.; Logue, Andrew R.; Tam, S. William; Tinker, William J.; Huang, Shiew-Mei; Chorvat, Robert J.Journal of Medicinal Chemistry (1998), 41 (23), 4615-4622CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)In an effort to improve the pharmacokinetic and pharmacodynamic properties of the cognition-enhancer linopirdine (DuP 996), a no. of core structure analogs were prepd. in which the 4-pyridyl pendant group was systematically replaced with 2-fluoro-4-pyridyl. This strategy resulted in the discovery of several compds. with improved activity in acetylcholine (ACh) release-enhancing assays, in vitro and in vivo. The most effective compd. resulting from these studies, 10,10-bis[(2-fluoro-4-pyridinyl)methyl]-9(10H)-anthracenone (I), is between 10 and 20 times more potent than linopirdine in increasing extracellular hippocampal ACh levels in the rat with a min. ED of 1 mg/kg. In addn. to superior potency, I possesses an improved pharmacokinetic profile compared to that of linopirdine. The half-life of I (2 h) in rats is 4-fold greater than that of linopirdine (0.5 h), and it showed a 6-fold improvement in brain-plasma distribution over linopirdine. On the basis of its pharmacol., pharmacokinetic, absorption, and distribution properties, I (DMP543) has been advanced for clin. evaluation as a potential palliative therapeutic for treatment of Alzheimer's disease.355Shaw, E.; Bernstein, J.; Losee, K.; Lott, W. A. Analogs of aspergillic acid. IV. Substituted 2-bromopyridine-N-oxides and their conversion to cyclic thiohydroxamic acids. J. Am. Chem. Soc. 1950, 72, 4362– 4364, DOI: 10.1021/ja01166a008[ACS Full Text], [CAS], Google Scholar355https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaG3MXis12qug%253D%253D&md5=f4f6deb3ae835708ce3a62213568875dAnalogs of aspergillic acid. IV. Substituted 2-bromopyridine N-oxides and their conversion to cyclic thiohydroxamic acidsShaw, Elliott; Bernstein, Jack; Losee, Kathryn; Lott, W. A.Journal of the American Chemical Society (1950), 72 (), 4362-4CODEN: JACSAT; ISSN:0002-7863.cf. C.A. 43, 8382g. Reaction of 1.0 mole 2-bromopyridine (I) and 1.2 moles BzO2H in CHCl3 4 days at room temp. and concn. of the 20% aq. HCl ext. gave 60% 2-bromopyridine 1-oxide-HCl (II), m. 135-6°. Derivs. of II prepd. similarly (read yield (%) and m.p.): 3-Me, 67, 179-80°; 4-Me, 59, 147-8°; 5-Me, 64, 141-2°; 6-Me, 61, 185-6°; 3-EtO, 52, 159-60°; 5-Br, 6.5, 165-6°. 2-Bromo-5-methylpyridine, prepd. in 77% yield, b14 97°, m. 42-3°; 6-Me isomer, b9 77°. Slow addn. of 1.5 moles 40% AcO2H to 1 mole I at 10-40°, heating 24 hrs. at 45-50°, concn. at 30° to 0.5 vol., addn. to ice and excess 40% KOH at 5°, CHCl3 extn., etc., gave 70% II. Neutralization of 0.31 mole II in 75 cc. H2O with 25% NaOH, portionwise addn. of 32 g. NaHSO3 in 150 cc. H2O at 100° during 1 hr., heating an addnl. 30 min. at 100°, cooling, and acidification of the filtrate with 6 N HCl yielded 61% 1-hydroxy-2(1H)-pyridinethione (III), m. 68-70°, gives a deep blue with FeCl3. III was also obtained from equimol. amts. of II and Na2S. The minimal inhibiting concns. of III in γ/cc. against Staphylococcus aureus P209, Klebsiella pneumoniae, and the bacillus of Calmette and Guerin were 0.06, 1.5, and 0.006, resp. Similar data for aspergillic acid were 20, 30, and 4 and for 1-hydroxy-2(1H)-pyridone (IV), 3, 40, and 2. Derivs. of III (read yield (%), m.p., and min. inhibiting concn.): 3-Me, 52, 74-5°, 0.06, 0.6, 0.004; 4-Me, 60, 59-61°, 0.08, 1.5, 0.001; 5-Me, 53, 106-7°, 0.07, 1.5, 0.001; 6-Me, 50%, 52-4°, 0.1, 3.5, 0.003; 3-EtO, 85, 101-3°, 0.08, 1.5, 0.03; 5-Br, 40%, 130-1°, 0.1, 2.0, 0.008. Refluxing II and 1 mole thiourea in abs. EtOH 1 hr. gave 72% 2-pyridyl-N-oxide-isothiourea HBr salt [2-(guanylmercapto)pyridine 1-oxide-HBr], m. 160-0.5° (decompn.), converted by 8% Na2CO3 in 4 hrs. at room temp. to 78% III. Heating III in 10% NaOH at 100° 1.5 hrs., concn. of the acidified soln. in vacuo, soln. of the residue in H2O, and addn. of aq. Cu(OAc)2 gave 53% Cu salt of IV. Warming 0.03 mole PhCH2SH, 0.012 mole II, and 0.043 Na in abs. EtOH 1 hr. at 50° letting stand 2 hrs. at room temp., addn. of excess NaOH, and EtOAc extn. gave 40% 2-(benzylmercapto)pyridine 1-oxide, m. 167-9°. BzO2H reacted quantitatively with 1 mole 2-benzylmercaptopyridine in CHCl3 to give 68% 2-pyridyl benzyl sulfoxide, m. 87-8°.356Sarantakis, D.; Sutherland, J. K.; Tortorella, C.; Tortorella, V. 2-Fluoropyridine N-oxide and its reactions with amino-acid derivatives. J. Chem. Soc. C 1968, 1, 72– 73, DOI: 10.1039/j39680000072357Chen, J.-G.; Markovitz, D. A.; Yang, A. Y.; Rabel, S. R.; Pang, J.; Dolinsky, O.; Wu, L.-S.; Alasandro, M. Degradation of a fluoropyridinyl drug in capsule formulation: degradant identification, proposed degradation mechanism, and formulation optimization. Pharm. Dev. Technol. 2000, 5, 561– 570, DOI: 10.1081/PDT-100102039[Crossref], [PubMed], [CAS], Google Scholar357https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXosV2nurc%253D&md5=aa625c3196719513f726a3dd5e2676a2Degradation of a fluoropyridinyl drug in capsule formulation: degradant identification, proposed degradation mechanism, and formulation optimizationChen, Jian-Ge; Markovitz, Debbie A.; Yang, Amy Y.; Rabel, Shelley R.; Pang, Josephine; Dolinsky, Olga; Wu, Lei-Shu; Alasandro, MarkPharmaceutical Development and Technology (2000), 5 (4), 561-570CODEN: PDTEFS; ISSN:1083-7450. (Marcel Dekker, Inc.)The purpose of this paper was to investigate the degrdn. chem. of a fluoropyridinyl drug candidate in capsule formulation and to optimize the formulation based on a proposed degrdn. mechanism. Small developmental batches of capsules were made by trituration of the drug and excipients by using a mortar and pestle, followed by manual encapsulation. Degradants were identified by LC-MS/MS and LC-photodiode array detector (PDA) and were monitored by LC-UV detector (UVD) during stability studies. The drug could undergo a nucleophilic substitution reaction in which hydroxyl groups replace the fluorine substituents on the pyridine rings. The initial degrdn. rate is independent of the drug concn. but dependent on the temp., the pH of the microenvironment, and the excipient type. On the basis of these exptl. results, a nucleophilic substitution reaction mechanism for the degrdn. was proposed and a successful capsule formulation was developed.358Rabel, S. R.; Shinwari, M. K.; Nemeth, G. A.; Blom, K. F.; Maurin, M. B. Characterization of the solution stability and degradation products of the nover neurotransmitter release enhancer 10,10-bis(2-fluoro-4-pyridinylmethyl)-9(10H)-anthracenone. Drug Stability 1997, 1, 224– 230[CAS], Google Scholar358https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXnvFKqtL0%253D&md5=46005c211cec07c5c63047a040a3e589Characterization of the solution stability and degradation products of the novel neurotransmitter release enhancer 10,10-bis(2-fluoro-4-pyridinylmethyl)-9(10H)-anthracenone (DMP 543)Rabel, Shelley R.; Shinwari, Mirwais K.; Nemeth, Gregory A.; Blom, Karl F.; Maurin, Michael B.Drug Stability (1997), 1 (4), 224-230CODEN: DRSTFY; ISSN:1355-5618. (Radcliffe Medical Press)The soln. stability of a neurotransmitter release enhancer, (10,10-bis(2-fluoro-4-pyridinylmethyl)-9(10H)-anthracenone) (DMP 543), was examd. as a function of pH and buffer concn. at 80°, μ = 0.5. DMP 543 underwent general and specific acid catalysis below pH 7, resulting in 2 major degrdn. products, while the compd. was stable at pH 7-9 after 51 days at 80°. The degradants were isolated by semi-preparative HPLC and characterized by proton, carbon and fluorine NMR and mass spectrometry. The major route of degrdn. was the sequential hydrolysis of the pyridine rings on the mol. to form the 2-hydroxy substituted products.359Snyder, G. L.; Prickaerts, J.; Wadenberg, M. L.; Zhang, L.; Zheng, H.; Yao, W.; Akkerman, S.; Zhu, H.; Hendrick, J. P.; Vanover, K. E.; Davis, R.; Li, P.; Mates, S.; Wennogle, L. P. Preclinical profile of ITI-214, an inhibitor of phosphodiesterase 1, for enhancement of memory performance in rats. Psychopharmacology (Berl.) 2016, 233, 3113– 3124, DOI: 10.1007/s00213-016-4346-2[Crossref], [PubMed], [CAS], Google Scholar359https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2s7jslaksA%253D%253D&md5=6fda37a7d71613a653ddebb26d8df313Preclinical profile of ITI-214, an inhibitor of phosphodiesterase 1, for enhancement of memory performance in ratsSnyder Gretchen L; Zhang Lei; Zheng Hailin; Yao Wei; Hendrick Joseph P; Vanover Kimberly E; Davis Robert; Li Peng; Mates Sharon; Wennogle Lawrence P; Prickaerts Jos; Akkerman Sven; Wadenberg Marie-Louise; Zhu HongwenPsychopharmacology (2016), 233 (17), 3113-24 ISSN:.RATIONALE: Therapeutic agents for memory enhancement in psychiatric disorders, such as schizophrenia, are urgently needed. OBJECTIVE: The aim of this study is to characterize the preclinical profile of ITI-214, a potent inhibitor of phosphodiesterase 1 (PDE1). METHODS: ITI-214 was assayed for inhibition of PDE1 versus other PDE enzyme families using recombinant human PDE enzymes and for off-target binding to 70 substrates (General SEP II diversity panel; Caliper Life Sciences). Effects of ITI-214 (0.1-10 mg/kg, po) on memory performance were assayed in rats using the novel object recognition (NOR) paradigm, with drug given at specified time points prior to or following exposure to objects in an open field. ITI-214 was evaluated for potential drug-drug interaction with risperidone in rats using conditioned avoidance response (CAR) and pharmacokinetic assessments. RESULTS: ITI-214 inhibited PDE1A (K i = 33 pmol) with >1000-fold selectivity for the nearest other PDE family (PDE4D) and displayed minimal off-target binding interactions in a 70-substrate selectivity profile. By using specific timing of oral ITI-214 administration, it was demonstrated in the NOR that ITI-214 is able to enhance acquisition, consolidation, and retrieval memory processes. All memory effects were in the absence of effects on exploratory behavior. ITI-214 did not disrupt the risperidone pharmacokinetic profile or effects in CAR. CONCLUSIONS: ITI-214 improved the memory processes of acquisition, consolidation, and retrieval across a broad dose range (0.1-10 mg/kg, po) without disrupting the antipsychotic-like activity of a clinical antipsychotic medication, specifically risperidone. Clinical development of ITI-214 is currently in progress.360Lee, Y. S.; Chuang, S. H.; Huang, L. Y.; Lai, C. L.; Lin, Y. H.; Yang, J. Y.; Liu, C. W.; Yang, S. C.; Lin, H. S.; Chang, C. C.; Lai, J. Y.; Jian, P. S.; Lam, K.; Chang, J. M.; Lau, J. Y.; Huang, J. J. Discovery of 4-aryl-N-arylcarbonyl-2-aminothiazoles as Hec1/Nek2 inhibitors. Part I: optimization of in vitro potencies and pharmacokinetic properties. J. Med. Chem. 2014, 57, 4098– 4110, DOI: 10.1021/jm401990s[ACS Full Text], [CAS], Google Scholar360https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXntFChtLw%253D&md5=e583b657fa4837f99545e596f08b6168Discovery of 4-Aryl-N-arylcarbonyl-2-aminothiazoles as Hec1/Nek2 Inhibitors. Part I: Optimization of in Vitro Potencies and Pharmacokinetic PropertiesLee, Ying-Shuan E.; Chuang, Shih-Hsien; Huang, Lynn Y. L.; Lai, Chun-Liang; Lin, Yu-Hsiang; Yang, Ju-Ying; Liu, Chia-Wei; Yang, Sheng-chuan; Lin, Her-Sheng; Chang, Chia-chi; Lai, Jun-Yu; Jian, Pei-Shiou; Lam, King; Chang, Jia-Ming; Lau, Johnson Y. N.; Huang, Jiann-JyhJournal of Medicinal Chemistry (2014), 57 (10), 4098-4110CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of 4-aryl-N-arylcarbonyl-2-aminothiazoles was designed and synthesized as Hec1/Nek2 inhibitors. Structural optimization led to compd. I bearing C-4' 4-methoxyphenoxy and 4-(o-fluoropyridyl)carbonyl groups that showed low nanomolar in vitro antiproliferative activity (IC50: 16.3-42.7 nM), high i.v. AUC (64.9 μM·h, 2.0 mg/kg) in SD rats, and significant in vivo antitumor activity (T/C = 32%, 20 mg/kg, IV) in mice bearing human MDA-MB-231 xenografts. Cell responses resulting from Hec1/Nek2 inhibition were obsd. in cells treated with I, including a reduced level of Hec1 coimmunopptd. with Nek2, degrdn. of Nek2, mitotic abnormalities, and apoptosis. Compd. I showed selectivity toward cancer cells over normal phenotype cells and was inactive in a [3H]astemizole competitive binding assay for hERG liability screening. Therefore, I is as a good lead toward the discovery of a preclin. candidate targeting Hec1/Nek2 interaction.361Johnson, C. M.; Linsky, T. W.; Yoon, D. W.; Person, M. D.; Fast, W. Discovery of halopyridines as quiescent affinity labels: inactivation of dimethylarginine dimethylaminohydrolase. J. Am. Chem. Soc. 2011, 133, 1553– 1562, DOI: 10.1021/ja109207m[ACS Full Text], [CAS], Google Scholar361https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjvFGgtA%253D%253D&md5=edfd01568d3bc3dca9d3f017cdc27cc4Discovery of Halopyridines as Quiescent Affinity Labels: Inactivation of Dimethylarginine DimethylaminohydrolaseJohnson, Corey M.; Linsky, Thomas W.; Yoon, Dae-Wi; Person, Maria D.; Fast, WalterJournal of the American Chemical Society (2011), 133 (5), 1553-1562CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)In an effort to develop novel covalent modifiers of dimethylarginine dimethylaminohydrolase (DDAH) that are useful for biol. applications, a set of "fragment"-sized inhibitors that were identified using a high-throughput screen are tested for time-dependent inhibition. One structural class of inactivators, 4-halopyridines, show time- and concn.-dependent inactivation of DDAH, and the inactivation mechanism of one example, 4-bromo-2-methylpyridine (1), is characterized in detail. The neutral form of halopyridines is not very reactive with excess glutathione. However, 1 readily reacts, with loss of its halide, in a selective, covalent, and irreversible manner with the active-site Cys249 of DDAH. This active-site Cys is not particularly reactive (pKa ca. 8.8), and 1 does not inactivate papain (Cys pKa ca. ≤4), suggesting that, unlike many reagents, Cys nucleophilicity is not a predominating factor in selectivity. Rather, binding and stabilization of the more reactive pyridinium form of the inactivator by a second moiety, Asp66, is required for facile reaction. This constraint imparts a unique selectivity profile to these inactivators. To our knowledge, halopyridines have not previously been reported as protein modifiers, and therefore they represent a first-in-class example of a novel type of quiescent affinity label.362Johnson, C. M.; Monzingo, A. F.; Ke, Z.; Yoon, D. W.; Linsky, T. W.; Guo, H.; Robertus, J. D.; Fast, W. On the mechanism of dimethylarginine dimethylaminohydrolase inactivation by 4-halopyridines. J. Am. Chem. Soc. 2011, 133, 10951– 10959, DOI: 10.1021/ja2033684[ACS Full Text], [CAS], Google Scholar362https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnvFagsrg%253D&md5=1c51b818e7ab51bb57831412e317c533On the Mechanism of Dimethylarginine Dimethylaminohydrolase Inactivation by 4-HalopyridinesJohnson, Corey M.; Monzingo, Arthur F.; Ke, Zhihong; Yoon, Dae-Wi; Linsky, Thomas W.; Guo, Hua; Robertus, Jon D.; Fast, WalterJournal of the American Chemical Society (2011), 133 (28), 10951-10959CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Small mols. capable of selective covalent protein modification are of significant interest for the development of biol. probes and therapeutics. We recently reported that 2-methyl-4-bromopyridine is a quiescent affinity label for the nitric oxide controlling enzyme dimethylarginine dimethylaminohydrolase (DDAH). Discovery of this novel protein modifier raised the possibility that the 4-halopyridine motif may be suitable for wider application. Therefore, the inactivation mechanism of the related compd. 2-hydroxymethyl-4-chloropyridine is probed here in more detail. Soln. studies support an inactivation mechanism in which the active site Asp66 residue stabilizes the pyridinium form of the inactivator, which has enhanced reactivity toward the active site Cys, resulting in covalent bond formation, loss of the halide, and irreversible inactivation. A 2.18 Å resoln. X-ray crystal structure of the inactivated complex elucidates the orientation of the inactivator and its covalent attachment to the active site Cys, but the structural model does not show an interaction between the inactivator and Asp66. Mol. modeling is used to investigate inactivator binding, reaction, and also a final pyridinium deprotonation step that accounts for the apparent differences between the soln.-based and structural studies with respect to the role of Asp66. This work integrates multiple approaches to elucidate the inactivation mechanism of a novel 4-halopyridine "warhead," emphasizing the strategy of using pyridinium formation as a "switch" to enhance reactivity when bound to the target protein.363Madhura, D. B.; Liu, J.; Meibohm, B.; Lee, R. E. Phase II metabolic pathways of spectinamide antitubercular agents: a comparative study of the reactivity of 4-substituted pyridines to glutathione conjugation. MedChemComm 2016, 7, 114– 117, DOI: 10.1039/C5MD00349K[Crossref], [PubMed], [CAS], Google Scholar363https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVGisrrE&md5=1c280109f629d3a6c6300d036fabc874Phase II metabolic pathways of spectinamide antitubercular agents: a comparative study of the reactivity of 4-substituted pyridines to glutathione conjugationMadhura, Dora B.; Liu, Jiuyu; Meibohm, Bernd; Lee, Richard E.MedChemComm (2016), 7 (1), 114-117CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)Spectinamides are promising new semisynthetic anti-tubercular agents that are modified with a pyridyl side chain, which blocks native efflux from the tuberculosis cell. This study, describes the stability of an advanced panel of spectinamide analogs, with varying substitutions to the pyridyl side chain, to Phase-II conjugative metab. by glucuronosyl transferase, sulfotransferase and glutathione-S-transferase enzymes using both human and rat S9 enzyme fractions. All solely 5-substituted pyridyl spectinamides exhibited complete stability towards Phase II conjugative enzymes. However, 4-chloro substituted pyridyl spectinamides were susceptible to glutathione conjugation with rates dependent on other substitutions to the pyridine ring. Electron donating 5-substitutions increased the propensity for glutathione conjugation and conversely the introduction of an electron withdrawing 5-fluoro group blocked all obsd. glutathione conjugation. Based on these Phase II metab. studies, lead spectinamides 1329, 1445, 1599, 1661 and 1810 were found to have favorable properties for potential lead compds. with no Phase II liabilities.364Okamura, T.; Kikuchi, T.; Okada, M.; Toramatsu, C.; Fukushi, K.; Takei, M.; Irie, T. Noninvasive and quantitative assessment of the function of multidrug resistance-associated protein 1 in the living brain. J. Cereb. Blood Flow Metab. 2009, 29, 504– 511, DOI: 10.1038/jcbfm.2008.135[Crossref], [PubMed], [CAS], Google Scholar364https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXisVSnsr8%253D&md5=0567e4c206f6481c8375f4e016f0f2b5Noninvasive and quantitative assessment of the function of multidrug resistance-associated protein 1 in the living brainOkamura, Toshimitsu; Kikuchi, Tatsuya; Okada, Maki; Toramatsu, Chie; Fukushi, Kiyoshi; Takei, Makoto; Irie, ToshiakiJournal of Cerebral Blood Flow & Metabolism (2009), 29 (3), 504-511CODEN: JCBMDN; ISSN:0271-678X. (Nature Publishing Group)Multidrug resistance-assocd. protein 1 (MRP1) acts as a defense mechanism by pumping xenobiotics and endogenous metabolites out of the brain. The currently available techniques for studying brain-to-blood efflux have significant limitations related to either their invasiveness or the qual. assessment. Here, we describe an in vivo method, which overcomes these limitations for assessing MRP1 function, using positron emission tomog. (PET) and a PET probe. 6-Bromo-7-[11C]methylpurine was designed to readily enter the brain after i.v. administration and to be efficiently converted to its glutathione conjugate (MRP1 substrate) in situ. Dynamic PET scan provided the brain time-activity curve after injection of 6-bromo-7-[11C]methylpurine into mice. The efflux rate of the substrate was kinetically estd. to be 1.4 h-1 with high precision. Moreover, knockout of Mrp1 gene caused approx. a 90% redn. of the efflux rate, compared with wild-type mice. In conclusion, our method allows noninvasive and quant. assessment for MRP1 function in the living brain.365Okamura, T.; Kikuchi, T.; Fukushi, K.; Arano, Y.; Irie, T. A novel noninvasive method for assessing glutathione-conjugate efflux systems in the brain. Bioorg. Med. Chem. 2007, 15, 3127– 3133, DOI: 10.1016/j.bmc.2007.02.045[Crossref], [PubMed], [CAS], Google Scholar365https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjslGntL4%253D&md5=b23e187ad43e3205bd8522757b0a4983A novel noninvasive method for assessing glutathione-conjugate efflux systems in the brainOkamura, Toshimitsu; Kikuchi, Tatsuya; Fukushi, Kiyoshi; Arano, Yasushi; Irie, ToshiakiBioorganic & Medicinal Chemistry (2007), 15 (9), 3127-3133CODEN: BMECEP; ISSN:0968-0896. (Elsevier Ltd.)Brain efflux systems export such conjugated metabolites as glutathione (GSH) and glucuronate conjugates, generated by the detoxification process, from the brain and serve to protect the brain from harmful metabolites. The intracerebral injection of a radiolabeled conjugate is a useful technique to assess brain efflux systems; however, this technique is not applicable to humans. Hence, we devised a novel noninvasive approach for assessing GSH-conjugate efflux systems using positron emission tomog. Here, we investigated whether or not a designed proprobe can deliver its GSH conjugate into the brain. Radiolabeled 6-chloro-7-methylpurine (7m6CP) was designed as the proprobe, and [14C]7m6CP was prepd. by the reaction of 6-chloropurine with [14C]CH3I as a model of [11C]CH3I. The radiochem. yield and purity of [14C]7m6CP were 10-20% and greater than 99%, resp. High brain uptake (0.8% ID/g) at 1 min was obsd., followed by gradual radioactivity clearance from the brain for 5-60 min after the injection of [14C]7m6CP into rats. Anal. of metabolites confirmed that the presence of [14C]7m6CP was hardly obsd., and 80% of the radioactivity was identical to its GSH conjugate for 15-60 min. The brain radioactivity was single-exponentially decreased during the period of 15-60 min post-injection of [14C]7m6CP, and the first-order efflux rate const. of the conjugate, estd. from the slope, was 0.0253 min-1. These results showed that (1) [14C]7m6CP readily entered the brain, (2) it efficiently and specifically transformed to the GSH conjugate within the brain, and (3) after [14C]7m6CP disappearance, the clearance of radioactivity represented the only efflux of GSH conjugate. We conclude that 7m6CP can deliver the GSH conjugate into the brain and would be useful for assessing GSH-conjugate efflux systems noninvasively.366Okamura, T.; Kikuchi, T.; Fukushi, K.; Irie, T. Reactivity of 6-halopurine analogs with glutathione as a radiotracer for assessing function of multidrug resistance-associated protein 1. J. Med. Chem. 2009, 52, 7284– 7288, DOI: 10.1021/jm901332c[ACS Full Text], [CAS], Google Scholar366https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlWmurfE&md5=7ebcc20c09d144e2e5df3c8b595362b7Reactivity of 6-Halopurine Analogs with Glutathione as a Radiotracer for Assessing Function of Multidrug Resistance-Associated Protein 1Okamura, Toshimitsu; Kikuchi, Tatsuya; Fukushi, Kiyoshi; Irie, ToshiakiJournal of Medicinal Chemistry (2009), 52 (22), 7284-7288CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)6-Bromo-7-[11C]methylpurine is reported to react with glutathione via glutathione S-transferases in the brain and to be converted into a substrate for multidrug resistance-assocd. protein 1 (MRP1), an efflux pump. The compd. with a rapid conversion rate allows quant. assessment of MRP1 function, but this rate is probably susceptible to interspecies differences. Hence, for application to different species, including humans, it is necessary to adjust the conversion rate by modifying the chem. structure. We therefore designed 6-halo-9-(or 7)-[ 14C]methylpurine (halogen: F, Cl, Br, or I), and evaluated them in vitro with respect to enzymic reactivity with glutathione using brain homogenates from the mouse, rat, or monkey. There was a marked difference in reactivity between these species. Changes in the position of the Me group and halogen on N-methyl-6-halopurine provided various compds. possessing wide-ranging reactivity with glutathione. In conclusion, the adjustment of reactivity of 6-bromo-7-[11C]methylpurine may allow assessment of MRP1 function in the brain in various species.367Liu, J.; Robins, M. J. S(N)Ar displacements with 6-(fluoro, chloro, bromo, iodo, and alkylsulfonyl)purine nucleosides: synthesis, kinetics, and mechanism1. J. Am. Chem. Soc. 2007, 129, 5962– 5968, DOI: 10.1021/ja070021u[ACS Full Text], [CAS], Google Scholar367https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXkt1ShsL0%253D&md5=2da27fced83f8cc103006f056109de56SNAr Displacements with 6-(Fluoro, Chloro, Bromo, Iodo, and Alkylsulfonyl)purine Nucleosides: Synthesis, Kinetics, and MechanismLiu, Jiangqiong; Robins, Morris J.Journal of the American Chemical Society (2007), 129 (18), 5962-5968CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)SNAr reactions with 6-(fluoro, chloro, bromo, iodo, and alkylsulfonyl)purine nucleosides and nitrogen, oxygen, and sulfur nucleophiles were studied. Pseudo-first-order kinetics were measured with 6-halopurine compds., and comparative reactivities were detd. vs. a 6-(alkylsulfonyl)purine nucleoside. The displacement reactivity order was: F > Br > Cl > I (with BuNH2/MeCN), F > Cl ≈ Br > I (with MeOH/1,8-diazabicyclo[5.4.0]undec-7-ene/MeCN), and F > Br > I > Cl [with KSCOCH3/DMSO]. The order of reactivity with a weakly basic arylamine (aniline) was: I > Br > Cl » F (with 5 equiv of aniline in MeCN at 70 °C). However, those reactions with aniline were auto-catalytic and had significant induction periods (∼50 min for the iodo compd. and ∼6 h for the fluoro analog). Addn. of trifluoroacetic acid (TFA) eliminated the induction period, and the order then was F > I > Br > Cl (with 5 equiv of aniline and 2 equiv of TFA in MeCN at 50 °C). The 6-(alkylsulfonyl)purine nucleoside analog was more reactive than the 6-fluoropurine compd. with both MeOH/DBU/MeCN and iPentSH/DBU/MeCN and was more reactive than the Cl, Br, and I compds. with BuNH2 and aniline/TFA. Titrn. of the 6-halopurine nucleosides in CDCl3 with TFA showed progressive downfield 1H NMR chem. shifts for H8 (larger) and H2 (smaller). The major site of protonation as N7 for both the 6-fluoro and 6-bromo analogs was confirmed by large upfield shifts (∼16 ppm) of the 15N NMR signal for N7 upon addn. of TFA (1.6 equiv). Mechanistic considerations and resoln. of prior conflicting results are presented.368Bradamante, S.; Pagani, G. A. Benzyl and heteroarylmethyl carbanions: structure and substituent effects. Adv. Carbanion Chem. 1997, 2, 189– 263, DOI: 10.1016/S1068-7394(96)80006-6369Abbotto, A.; Alanzo, V.; Bradamante, S.; Pagani, G. A. Preparation of heteroaryl phenylmethanes and a 13C and 15N NMR spectroscopic study of their conjugate carbanions. Rotational isomerism and charge maps of the anions and ranking of the charge demands of the heterocycles. J. Chem. Soc., Perkin Trans. 2 1991, 481– 488, DOI: 10.1039/p29910000481[Crossref], [CAS], Google Scholar369https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXktVOqtb0%253D&md5=8e6fc4be2b709d75303979f360d42a8dPreparation of heteroaryl phenylmethanes and a carbon-13 and nitrogen-15 NMR spectroscopic study of their conjugate carbanions. Rotational isomerism and charge maps of the anions and ranking of the charge demands of the heterocyclesAbbotto, Alessandro; Alanzo, Vito; Bradamante, Silvia; Pagani, Giorgio A.Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry (1972-1999) (1991), (4), 481-8CODEN: JCPKBH; ISSN:0300-9580.2-Benzylpyridazine, 4-benzylpyrimidine, 2-benzylpyrimidine and 2-benzylpyrazine have been prepd. in order to study their 13C and 15N spectra and those of their conjugate carbanions. These systems are aza-homologs of the previously reported benzylpyridines and have been considered in order to evaluate the effect of aza-substitution upon rotational isomerism and charge maps in the anions. Two synthetic approaches have been followed: (i) decarboxylation of α-(heteroaryl)phenylacetic acids, in turn obtained by nucleophilic substitution of phenylacetonitrile anion on the pertinent haloazine (or a correspondingly available deriv.); (ii) by nucleophilic substitution of benzyl(tributylphosphonium)ylide on the pertinent haloazine. The 13C and 15N NMR data for the conjugate carbanions indicate that, at room temp., there is slow rotation about the bond between the carbanionic carbon and the carbon atom of the heterocycle: this generates geometrical isomerism in the anions. The NMR data are treated with the π-charge-shift equations to obtain the local variations of the π-electron d. In the anions, from the fraction of π-charge transferred to the heterocycles, it is possible to obtain the charge demands of the heterocycles and thus rank them on the same scale as primary org. functionalities. 4-Pyrimidyl is the strongest electron-withdrawing heterocyclic residue, comparable with the acetyl group.370Shirasaka, T.; Murakami, K.; Ford, H., Jr.; Kelley, J. A.; Yoshioka, H.; Kojima, E.; Aoki, S.; Broder, S.; Mitsuya, H. Lipophilic halogenated congeners of 2’,3′-dideoxypurine nucleosides active against human immunodeficiency virus in vitro. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 9426– 9430, DOI: 10.1073/pnas.87.23.9426[Crossref], [PubMed], [CAS], Google Scholar370https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXpsVKguw%253D%253D&md5=1c73ce3c59782599733ee83429b9333aLipophilic halogenated congeners of 2',3'-dideoxypurine nucleosides active against human immunodeficiency virus in vitroShirasaka, Takuma; Murakami, Kunichika; Ford, Harry, Jr.; Kelley, James A.; Yoshioka, Hidetoshi; Kojima, Eiji; Aoki, Shizuko; Broder, Samuel; Mitsuya, HiroakiProceedings of the National Academy of Sciences of the United States of America (1990), 87 (23), 9426-30CODEN: PNASA6; ISSN:0027-8424.Four 2-amino-6-halo- and four 6-halo-2',3'-dideoxypurine ribofuranosides (ddPs) I (R1 = Cl, F, Br, I, SH; R2 = H or NH2) tested for in vitro activity to suppress the infectivity, cytopathic effect, Gag protein expression, and DNA synthesis of human immunodeficiency virus (HIV). The comparative order of in vitro anti-HIV activity of the eight 6-halo-ddPs was as follows: 2-amino-6-fluoro, 2-amino-6-chloro, 6-fluoro > 2-amino-6-bromo > 2-amino-6-iodo, 6-chloro > 6-bromo > 6-iodo. 2-Amino-6-fluoro-, 2-amino-6-chloro-, and 6-fluoro-ddPs showed a potent activity against HIV comparable to that of 2',3'-dideoxyinosine (ddI) or 2',3'-dideoxyguanosine (ddG) and completely blocked the infectivity of HIV without affecting the growth of target cells. The lipophilciity order was as follows: 2-amino-6-iodo > 2-amino-6-bromo > 2-amino-6-chloro > 2-amino-6-fluoro » ddG > ddI. All eight 6-halo-ddPs were substrates for adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4). The relative rates of hydrolysis by ADA were as follows: ddA, 2-amino-6-fluoro » 2-amino-6-chloro, 2-amino-6-bromo > 2-amino-6-iodo. Taken together, these compds. may represent an addnl. class of lipophilic prodrugs for ddI and ddG and may also provide a strategy for endowing therapeutic purine nucleosides with desirable lipophilicity.371Morgan, M. E.; Chi, S. C.; Murakami, K.; Mitsuya, H.; Anderson, B. D. Central nervous system targeting of 2’,3′-dideoxyinosine via adenosine deaminase-activated 6-halo-dideoxypurine prodrugs. Antimicrob. Agents Chemother. 1992, 36, 2156– 2165, DOI: 10.1128/AAC.36.10.2156[Crossref], [PubMed], [CAS], Google Scholar371https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XmtF2qsbw%253D&md5=48627e036850f263d2013982faf0dacfCentral nervous system targeting of 2',3'-dideoxyinosine via adenosine deaminase-activated 6-halo-dideoxypurine prodrugsMorgan, M. E.; Chi, S. C.; Murakami, K.; Mitsuya, H.; Anderson, B. D.Antimicrobial Agents and Chemotherapy (1992), 36 (10), 2156-65CODEN: AMACCQ; ISSN:0066-4804.AIDS dementia complex is a neurol. disorder, characterized by increasingly severe cognitive, behavioral, and motor impairment, which is assocd. with human immunodeficiency virus (HIV) infection in the central nervous system (CNS). Many of the dideoxynucleosides effective systemically in the treatment of HIV infections, such as 2',3'-dideoxyinosine (ddI), exhibit limited penetration into the CNS and limited or variable effectiveness in reversing the symptoms of AIDS dementia. Thus, approaches for increasing the CNS uptake of ddI and other dideoxynucleosides are needed. The CNS uptake of a series of 6-halo-2',3'-dideoxypurine ribofuranosides (6-halo-ddPs) previously shown to be active against HIV because of their conversion to ddI through the action of adenosine deaminase was examd. in rats. In vitro studies in rat blood and brain tissue homogenate suggested a favorable selectivity for bioconversion in brain tissue, but with bioconversion half-lives varying widely within the series. In vivo infusions of 6-chloro-ddP (6-Cl-ddP), 6-bromo-ddP (6-Br-ddP), and 6-iodo-ddP (6-I-ddP) resulted in significant increases (20- to 34-fold) in the ddI concn. ratios in brain parenchyma/plasma when compared with those after an infusion of ddI alone. Abs. concns. of ddI in brain parenchyma were increased 10- and 4-fold, resp., following 30-min infusions of 6-Cl-ddP or 6-Br-ddP, but were 2.4 fold lower after an infusion of 6-I-ddP relative to that after a control infusion of ddI. Detailed studies of the plasma pharmacokinetics, CNS uptake kinetics, and bioconversion of 6-Cl-ddP were conducted to compare in vivo transport and bioconversion parameters with those predicted from in vitro measurements and to rationalize the efficiency of CNS delivery of ddI from 6-Cl-ddP. The results show that increased lipophilicity alone does not ensure that a given prodrug will deliver higher levels of a parent compd. to the CNS. Both the selectivity and abs. rate of bioconversion in the brain are important factors.372Kim, D.-K.; Kim, J.-K.; Chae, Y.-B. Design and synthesis of 6-fluoropurine acyclonucleosides: potential prodrugs of acyclovir and ganciclovir. Bioorg. Med. Chem. Lett. 1994, 4, 1309– 1312, DOI: 10.1016/S0960-894X(01)80350-4[Crossref], [CAS], Google Scholar372https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXmtVGrtL0%253D&md5=9478dbb3766d5fcb5c0fa8f977c15e9bDesign and synthesis of 6-fluoropurine acyclonucleosides: potential prodrugs of acyclovir and ganciclovirKim, Dae Kee; Kim, Hee Kap; Chae, Young BokBioorganic & Medicinal Chemistry Letters (1994), 4 (11), 1309-12CODEN: BMCLE8; ISSN:0960-894X.6-Fluoropurine acyclic nucleosides I (R = H, CH2OH, R1 = F) (II) have been prepd. as potential prodrugs of acyclovir and ganciclovir. It has been found that II are 11.6 and 7.6 times more efficiently metabolized to acyclovir and ganciclovir by adenosine deaminase than the corresponding 6-aminopurine acyclonucleosides I (R = H, CH2OH, R1 = NH2).373Kim, D. K.; Lee, N.; Im, G. J.; Kim, H. T.; Kim, K. H. Synthesis and evaluation of 2-amino-6-fluoro-9-(2-hydroxyethoxymethyl)purine esters as potential prodrugs of acyclovir. Bioorg. Med. Chem. 1998, 6, 2525– 2530, DOI: 10.1016/S0968-0896(98)80026-6[Crossref], [PubMed], [CAS], Google Scholar373https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXjvVKhsw%253D%253D&md5=417b0d155bab9c6fe31d8871160d87c6Synthesis and evaluation of 2-amino-6-fluoro-9-(2-hydroxyethoxymethyl)purine esters as potential prodrugs of acyclovirKim, Dae-Kee; Lee, Namkyu; Im, Guang-Jin; Kim, Hun-Taek; Kim, Key H.Bioorganic & Medicinal Chemistry (1998), 6 (12), 2525-2530CODEN: BMECEP; ISSN:0968-0896. (Elsevier Science Ltd.)2-Amino-6-fluoro-9-(2-hydroxyethoxymethyl)purine (I) and its ester derivs. were synthesized as potential prodrugs of acyclovir, and were evaluated for their oral acyclovir bioavailability in rats and in vivo antiviral efficacy in HSV-1-infected mice. Treatment of 2-amino-6-chloro-9-(2-hydroxyethoxymethyl)purine with trimethylamine in THF/DMF (4:1) followed by a reaction of the resulting trimethylammonium chloride salt with KF in DMF gave I in 78% yield. Esterification of I with an appropriate acid anhydride (Ac2O, (EtCO)2O, (n-PrCO)2O, or (i-PrCO)2O) in DMF in the presence of a catalytic amt. of DMAP at room temp. produced the esters in 90-98% yields. Of the prodrugs tested in rats, the isobutyrate achieved the highest mean urinary recovery of acyclovir (51%) that is 5.7-fold higher than that of acyclovir (9%) and comparable to that of valacyclovir (50%). The prodrug isobutyrate protected dose-dependently the mortality of HSV-1-infected mice, and the group treated with the isobutyrate at a dose of 400 mg/kg showed the longest mean survival day (14.6 ± 3.1 days) (mean ± S.D.).374Kim, D. K.; Chang, K.; Im, G. J.; Kim, H. T.; Lee, N.; Kim, K. H. Synthesis and evaluation of 2-amino-9-(1, 3-dihydroxy-2-propoxymethyl)- 6-fluoropurine mono- and diesters as potential prodrugs of ganciclovir. J. Med. Chem. 1999, 42, 324– 328, DOI: 10.1021/jm980321+[ACS Full Text], [CAS], Google Scholar374https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXos1Gi&md5=dbca4a20f373e22a7b32f86879198c96Synthesis and evaluation of 2-amino-9-(1,3-dihydroxy-2-propoxymethyl)- 6-fluoropurine mono- and diesters as potential prodrugs of ganciclovirKim, Dae-Kee; Chang, Kieyoung; Im, Guang-Jin; Kim, Hun-Taek; Lee, Namkyu; Kim, Key H.Journal of Medicinal Chemistry (1999), 42 (2), 324-328CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of 2-amino-9-(1,3-dihydroxy-2-propoxymethyl)-6-fluoropurine mono- and diesters, I [R = COMe, COEt, CO(CH2)2Me, COCHMe2, H, R1 = COMe, COEt, CO(CH2)2Me, COCHMe2], were synthesized as potential prodrugs of ganciclovir and evaluated for their oral ganciclovir bioavailability in rats. Treatment of 2-amino-6-chloro-9-(1,3-dihydroxy-2-propoxymethyl)purine with Me3N in DMF/THF (1/4) followed by the reaction of the resulting trimethylammonium chloride salt with KF in DMF gave 2-amino-9-(1,3-dihydroxy-2-propoxymethyl)-6-fluoropurine (II) in 83% yield. Esterification of II with an appropriate acid anhydride (Ac2O, (EtCO)2O, (n-PrCO)2O, or (i-PrCO)2O) in DMF in the presence of a catalytic amt. of DMAP produced the diesters in 92-98% yields and the monoesters in 37-44% yields. Of the prodrugs tested in rats, the monoisobutyrate achieved the highest ganciclovir bioavailability (45%) that is 15-fold higher than that from ganciclovir (3%), followed in order by the diisobutyrate (42%), the diacetate (41%), the monobutyrate (41%), the monopropionate (39%), the dipropionate (35%), the dibutyrate (35%), and the monoacetate (29%). The prodrugs I were found to be quite stable at pH 6.0 (t1/2 = >29 days), 7.4 (t1/2 = >7 days), and 8.0 (t1/2 = >2 days) but had relatively short half-lives at pH 1.2 (t1/2 = 60-83 min).375Kim, D. K.; Lee, N.; Kim, H. T.; Im, G. J.; Kim, K. H. Synthesis and evaluation of 2-amino-6-fluoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine mono- and diesters as potential prodrugs of penciclovir. Bioorg. Med. Chem. 1999, 7, 565– 570, DOI: 10.1016/S0968-0896(98)00263-6[Crossref], [PubMed], [CAS], Google Scholar375https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXitlGktbk%253D&md5=6620297f8c4cbc0724a273fa7197761fSynthesis and Evaluation of 2-amino-6-fluoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine mono- and diesters as potential prodrugs of penciclovirKim, Dae-Kee; Lee, Namkyu; Kim, Hun-Taek; Im, Guang-Jin; Kim, Key H.Bioorganic & Medicinal Chemistry (1999), 7 (3), 565-570CODEN: BMECEP; ISSN:0968-0896. (Elsevier Science Ltd.)2-Amino-6-fluoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine, and its mono- and diesters were prepd. and evaluated for their potential as prodrugs of penciclovir. Treatment of 2-amino-6-chloro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine with trimethylamine in THF followed by a reaction of the resulting trimethylammonium chloride salt with KF in DMF afforded 2-amino-6-fluoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine in 80% yield. Esterification with an appropriate acid anhydride [Ac2O, (EtCO)2O, (n-PrCO)2O, or (i-PrCO)2O] in DMF in the presence of a catalytic amt. of DMAP produced the mono-esters in 42-45% yields and diesters in 87-99% yields. Of the prodrugs tested in rats, the mono-isobutyrate was the most efficiently absorbed and metabolized, showing the mean max. total concn. of penciclovir (5.5 μg/mL) and 2-Amino-6-fluoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine (10.8 μg/mL) in the blood was much higher than the mean max. concn. of penciclovir (11.5 μg/mL) from famciclovir. However, the mean concns. of penciclovir from the mono-isobutyrate were lower than those from famciclovir because of the limited conversion of a major metabolite to penciclovir by adenosine deaminase.376Ouvry, G.; Clary, L.; Tomas, L.; Aurelly, M.; Bonnary, L.; Borde, M.; Bouix-Peter, C.; Chantalat, L.; Defoin-Platel, C.; Deret, S.; Forissier, M.; Harris, C. S.; Isabet, T.; Lamy, L.; Luzy, A.-P.; Pascau, J.; Soulet, C.; Taddei, A.; Taquet, N.; Thoreau, E.; Varvier, E.; Vial, E.; Hennequin, L. F. Impact of minor structural modifications on properties of a series of mTOR inhibitors. ACS Med. Chem. Lett. 2019, 10, 1561– 1567, DOI: 10.1021/acsmedchemlett.9b00401[ACS Full Text], [CAS], Google Scholar376https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvFSru7bI&md5=aa5bf253d605bbc6bfe1dfbf43de24b8Impact of Minor Structural Modifications on Properties of a Series of mTOR InhibitorsOuvry, Gilles; Clary, Laurence; Tomas, Loic; Aurelly, Michele; Bonnary, Laetitia; Borde, Emilie; Bouix-Peter, Claire; Chantalat, Laurent; Defoin-Platel, Claire; Deret, Sophie; Forissier, Mathieu; Harris, Craig S.; Isabet, Tatiana; Lamy, Laurent; Luzy, Anne-Pascale; Pascau, Jonathan; Soulet, Catherine; Taddei, Alessandro; Taquet, Nathalie; Thoreau, Etienne; Varvier, Emeric; Vial, Emmanuel; Hennequin, Laurent F.ACS Medicinal Chemistry Letters (2019), 10 (11), 1561-1567CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)Minor structural modifications (sometimes single atom changes) can have a dramatic impact on the properties of compds. This is illustrated here on structures related to known mTOR inhibitor Sapanisertib. Subtle changes in the hinge binder lead to strikingly different overall profiles with changes in phys. properties, metab., and kinase selectivity.377Boehringer, M.; Fischer, H.; Hennig, M.; Hunziker, D.; Huwyler, J.; Kuhn, B.; Loeffler, B. M.; Luebbers, T.; Mattei, P.; Narquizian, R.; Sebokova, E.; Sprecher, U.; Wessel, H. P. Aryl- and heteroaryl-substituted aminobenzo[a]quinolizines as dipeptidyl peptidase IV inhibitors. Bioorg. Med. Chem. Lett. 2010, 20, 1106– 1108, DOI: 10.1016/j.bmcl.2009.12.025[Crossref], [PubMed], [CAS], Google Scholar377https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVGrsb4%253D&md5=b1d3c1eaa70297eab5ca1abfcd1d7e5dAryl- and heteroaryl-substituted aminobenzo[a]quinolizines as dipeptidyl peptidase IV inhibitorsBoehringer, Markus; Fischer, Holger; Hennig, Michael; Hunziker, Daniel; Huwyler, Joerg; Kuhn, Bernd; Loeffler, Bernd M.; Luebbers, Thomas; Mattei, Patrizio; Narquizian, Robert; Sebokova, Elena; Sprecher, Urs; Wessel, Hans PeterBioorganic & Medicinal Chemistry Letters (2010), 20 (3), 1106-1108CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Synthesis and SAR are described for a structurally distinct class of DPP-IV inhibitors based on aminobenzo[a]quinolizines bearing (hetero-)arom. substituents in the S1 specificity pocket. The m-(fluoromethyl)-Ph deriv. (S,S,S)-I possesses the best fit in the S1 pocket. However, (S,S,S)-II, bearing a more hydrophilic 5-methyl-pyridin-2-yl residue as substituent for the S1 pocket, displays excellent in vivo activity and superior drug-like properties.378Mattei, P.; Boehringer, M.; Di Giorgio, P.; Fischer, H.; Hennig, M.; Huwyler, J.; Kocer, B.; Kuhn, B.; Loeffler, B. M.; Macdonald, A.; Narquizian, R.; Rauber, E.; Sebokova, E.; Sprecher, U. Discovery of carmegliptin: a potent and long-acting dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. Bioorg. Med. Chem. Lett. 2010, 20, 1109– 1113, DOI: 10.1016/j.bmcl.2009.12.024[Crossref], [PubMed], [CAS], Google Scholar378https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVGrsb8%253D&md5=448a62baa339f33ea89a9f7d862a7c25Discovery of carmegliptin: A potent and long-acting dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetesMattei, Patrizio; Boehringer, Markus; Di Giorgio, Patrick; Fischer, Holger; Hennig, Michael; Huwyler, Joerg; Kocer, Buelent; Kuhn, Bernd; Loeffler, Bernd M.; MacDonald, Alexander; Narquizian, Robert; Rauber, Etienne; Sebokova, Elena; Sprecher, UrsBioorganic & Medicinal Chemistry Letters (2010), 20 (3), 1109-1113CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Design, synthesis, and SAR are described for a class of DPP-IV inhibitors based on aminobenzo[a]quinolizines with non-arom. substituents in the S1 specificity pocket. One representative thereof, carmegliptin (I), was chosen for clin. development. The X-ray structure in complex of I with the enzyme and early efficacy data in animal models of type 2 diabetes are also presented.379Kuhlmann, O.; Paehler, A.; Weick, I.; Funk, C.; Pantze, M.; Zell, M.; Timm, U. Pharmacokinetics and metabolism of the dipeptidyl peptidase IV inhibitor carmegliptin in rats, dogs, and monkeys. Xenobiotica 2010, 40, 840– 852, DOI: 10.3109/00498254.2010.519406[Crossref], [PubMed], [CAS], Google Scholar379https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3cbls1ehsg%253D%253D&md5=41599b82f0246f119097a818d0a1c7abPharmacokinetics and metabolism of the dipeptidyl peptidase IV inhibitor carmegliptin in rats, dogs, and monkeysKuhlmann Olaf; Paehler Axel; Weick Idelette; Funk Christoph; Pantze Michael; Zell Manfred; Timm UweXenobiotica; the fate of foreign compounds in biological systems (2010), 40 (12), 840-52 ISSN:.The pharmacokinetics and excretion of carmegliptin, a novel dipeptidyl peptidase IV inhibitor, were examined in rats, dogs, and cynomolgus monkeys. Carmegliptin exhibited a moderate clearance, extensive tissue distribution, and a variable oral bioavailability of 28-174%. Due to saturation of intestinal active secretion, the area under the plasma concentration-time curve (AUC) in dogs and monkeys increased in a more than dose-proportional manner over an oral dose range of 2.5-10 mg/kg. Following oral administration of [(14)C]carmegliptin at 3 mg/kg, > 94% of the radioactive dose was recovered in 72-h post-dose from Wistar rats and Beagle dogs. Virtually, the entire administered radioactive dose was excreted unchanged in urine, intestinal lumen, and bile. Approximately 36%, 29%, and 19% of the dose were excreted by respective routes. Consistently, in vitro, carmegliptin was highly resistant to hepatic metabolism in all species tested. Based on in vitro studies, carmegliptin is a good substrate for Mdr1/MDR1. Breast cancer resistance protein (Bcrp) is not expected to be involved in the transport of carmegliptin since in vitro carmegliptin was not significantly transported by this transporter. The very high extravascular distribution of carmegliptin in the intestinal tissues, as demonstrated in Wistar rats and Beagle dogs, could play a significant role in its therapeutic effect.380Shetty, H. U.; Zoghbi, S. S.; Simeon, F. G.; Liow, J. S.; Brown, A. K.; Kannan, P.; Innis, R. B.; Pike, V. W. Radiodefluorination of 3-fluoro-5-(2-(2-[18F](fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile ([18F]SP203), a radioligand for imaging brain metabotropic glutamate subtype-5 receptors with positron emission tomography, occurs by glutathionylation in rat brain. J. Pharmacol. Exp. Ther. 2008, 327, 727– 735, DOI: 10.1124/jpet.108.143347[Crossref], [PubMed], [CAS], Google Scholar380https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVCjtLvN&md5=7cc78a76561a92270f956ca354d259efRadiodefluorination of 3-fluoro-5-(2-(2-[18F](fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile ([18F]SP203), a radioligand for imaging brain metabotropic glutamate subtype-5 receptors with positron emission tomography, occurs by glutathionylation in rat brainShetty, H. Umesha; Zoghbi, Sami S.; Simeon, Fabrice G.; Liow, Jeih-San; Brown, Amira K.; Kannan, Pavitra; Innis, Robert B.; Pike, Victor W.Journal of Pharmacology and Experimental Therapeutics (2008), 327 (3), 727-735CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)Metabotropic glutamate subtype-5 receptors (mGluR5) are implicated in several neuropsychiatric disorders. Positron emission tomog. (PET) with a suitable radioligand may enable monitoring of regional brain mGluR5 d. before and during treatments. We have developed a new radioligand, 3-fluoro-5-(2-(2-[18F](fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile ([18F]SP203), for imaging brain mGluR5 in monkey and human. In monkey, radioactivity was obsd. in bone, showing release of [18F]fluoride ion from [18F]SP203. This defluorination was not inhibited by disulfiram, a potent inhibitor of CYP2E1. PET confirmed bone uptake of radioactivity and therefore defluorination of [18F]SP203 in rats. To understand the biochem. basis for defluorination, we administered [18F]SP203 plus SP203 in rats for ex vivo anal. of metabolites. Radio-high-performance liq. chromatog. detected [18F]fluoride ion as a major radiometabolite in both brain ext. and urine. Incubation of [18F]SP203 with brain homogenate also generated this radiometabolite, whereas no metab. was detected in whole blood in vitro. Liq. chromatog.-mass spectrometry anal. of the brain ext. detected m/z 548 and 404 ions, assignable to the [M + H]+ of S-glutathione (SP203Glu) and N-acetyl-S-L-cysteine (SP203Nac) conjugates of SP203, resp. In urine, only the [M + H]+ of SP203Nac was detected. Mass spectrometry/mass spectrometry and multi-stage mass spectrometry analyses of each metabolite yielded product ions consistent with its proposed structure, including the former fluoromethyl group as the site of conjugation. Metabolite structures were confirmed by similar analyses of SP203Glu and SP203Nac, prepd. by glutathione S-transferase reaction and chem. synthesis, resp. Thus, glutathionylation at the 2-fluoromethyl group is responsible for the radiodefluorination of [18F]SP203 in rat. This study provides the first demonstration of glutathione-promoted radiodefluorination of a PET radioligand.381McDonald, I. A.; Nyce, P. L.; Jung, M. J.; Sabol, J. S. Syntheses of dl-2-fluoromethyl-p-tyrosine and dl-2-difluoromethyl-p-tyrosine as potential inhibitors of tyrosine hydroxylase. Tetrahedron Lett. 1991, 32, 887– 890, DOI: 10.1016/S0040-4039(00)92112-9[Crossref], [CAS], Google Scholar381https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXhvVKjtrY%253D&md5=94ea2a823059fc2697e102cefd0486d9Syntheses of DL-2-fluoromethyl-p-tyrosine and DL-2-(difluoromethyl)-p-tyrosine as potential inhibitors of tyrosine hydroxylaseMcDonald, Ian A.; Nyce, Philip L.; Jung, Michel J.; Sabol, Jeffrey S.Tetrahedron Letters (1991), 32 (7), 887-90CODEN: TELEAY; ISSN:0040-4039.The title compds. I (R = H and F) were prepd. from o-xylene II and benzoate III, resp. I (R = H) was obtained from II in 11 steps; a key step was the free radical bromination of tyrosine IV (Boc = Me3CO2C, R1 = H) with NBS followed by treatment with AgF to give IV (R1 = F). I (R = F) was prepd. from III in 11 steps. I (R = H, F) were partially characterized as competitive inhibitors of purified bovine adrenal tyrosine hydroxylase.382Robinson, C. Metyrosine. Drugs Today 1980, 16, 343– 348[CAS], Google Scholar382https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXntFagsA%253D%253D&md5=dac351a91b456574062cfc5f851e82f8MetyrosineRobinson, C.Drugs of Today (1980), 16 (10), 343-8CODEN: MDACAP; ISSN:0025-7656.The present status of the drug metyrosine (I) [672-87-7] is reviewed; 10 refs.383Woolridge, E. M.; Rokita, S. E. 6-(Difluoromethyl)tryptophan as a probe for substrate activation during the catalysis of tryptophanase. Biochemistry 1991, 30, 1852– 1857, DOI: 10.1021/bi00221a018[ACS Full Text], [CAS], Google Scholar383https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXnslKgtw%253D%253D&md5=672d34bfbe9d3dcd4d7f7772565472056-(Difluoromethyl)tryptophan as a probe for substrate activation during the catalysis of tryptophanaseWoolridge, Elisa M.; Rokita, Steven E.Biochemistry (1991), 30 (7), 1852-7CODEN: BICHAW; ISSN:0006-2960.A substrate analog, 6-(difluoromethyl)tryptophan, was developed and characterized for mechanistic investigation of tryptophanase of Escherichia coli. The utility of this deriv. was based on its ability to partition between F- elimination and C-C bond scission during tryptophan metab. The nonenzymic hydrolysis to 6-formyltryptophan occurred slowly under neutral conditions with a 1st-order rate const. of 0.0039 min-1. This process, however, was accelerated by 104-fold upon deprotonation of the indolyl N-1 atom at high pH. Tryptophanase did not detectably facilitate this hydrolysis reaction, since no protein-dependent conversion of the difluoromethyl group was detected. Instead, the enzyme accepted the fluorinated species as an analog of tryptophan and catalyzed the corresponding formation of 6-(difluoromethyl)indole, pyruvate, and NH4+. Anionic intermediates are therefore not expected to form during the catalytic activation of the indolyl moiety. Instead, arom. protonation likely promotes the release of indole during enzymic degrdn. of tryptophan.384Kai, H.; Hinou, H.; Nishimura, S. Aglycone-focused randomization of 2-difluoromethylphenyl-type sialoside suicide substrates for neuraminidases. Bioorg. Med. Chem. 2012, 20, 2739– 2746, DOI: 10.1016/j.bmc.2012.02.001[Crossref], [PubMed], [CAS], Google Scholar384https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xjs1Oqtbg%253D&md5=935f17620b75bcf8fffdbd1eee849ab7Aglycone-focused randomization of 2-difluoromethylphenyl-type sialoside suicide substrates for neuraminidasesKai, Hirokazu; Hinou, Hiroshi; Nishimura, Shin-IchiroBioorganic & Medicinal Chemistry (2012), 20 (8), 2739-2746CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)A selective and potent inhibitor of neuraminidases, a hydrolase that is responsible for processing sialylated glycoconjugates, is a promising drug candidate for various infective diseases. The current study demonstrates that the use of an aglycon-focused library of 2-difluoromethylphenyl α-sialosides is an effective technique to find potent and selective mechanism-based labeling reagents for neuraminidases. The focused library was constructed from a 4-azide-2-difluoromethylphenyl sialoside (2) and an alkyne-terminated compd. library by a click reaction. The focused library showed different inhibition patterns for two neuraminidases, Vibrio cholerae neuraminidase (VCNA) and human neuraminidase 2 (hNeu2), and the most potent inhibitors for each neuraminidase were selected. A kinetic anal. of the selected inhibitors demonstrated that the modification of the aglycon moiety improved the KI value with little change in the t1/2 value of the enzyme activity relative to the basic skeleton (2).385Kurogochi, M.; Nishimura, S.; Lee, Y. C. Mechanism-based fluorescent labeling of β-galactosidases. An efficient method in proteomics for glycoside hydrolases. J. Biol. Chem. 2004, 279, 44704– 44712, DOI: 10.1074/jbc.M401718200[Crossref], [PubMed], [CAS], Google Scholar385https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXosFymtLw%253D&md5=110f679fc05568ebe1bbac2a3db35bfcMechanism-based Fluorescent Labeling of β-Galactosidases: An Efficient Method in Proteomics for Glycoside HydrolasesKurogochi, Masaki; Nishimura, Shin-Ichiro; Lee, Yuan ChuanJournal of Biological Chemistry (2004), 279 (43), 44704-44712CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)(4-N-5-Dimethylaminonaphthalene-1-sulfonyl-2-difluoromethylphenyl)-β-D-galactopyranoside was synthesized and successfully tested on β-galactosidases from Xanthomonas manihotis, Escherichia coli, and Bacillus circulans for the rapid identification of the catalytic site. Reaction of the irreversible inhibitor with enzymes proceeded to afford a fluorescence-labeled protein suitable for further high throughput characterization by using antidansyl antibody and matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF). Specific probing by a fluorescent aglycon greatly facilitated identification of the labeled peptide fragments from β-galactosidases. It was demonstrated by using X. manihotis β-galactosidase that the Arg-58 residue, which is located within a sequence of 56IPRAYWKD63, was labeled by nucleophilic attack of the guanidinyl group. This sequence including Arg-58 (Leu-46 to Tyr-194) was similar to that (Met-1 to Tyr-151) of Thermus thermophilus A4, which is the first known structure of glycoside hydrolases family 42. A catalytic glutamic acid (Glu-537) of E. coli β-galactosidase was shown to be labeled by the same procedure, suggesting that the modification site with this irreversible substrate might depend both on the nucleophilicity of the amino acids and their spatial arrangement in the individual catalytic cavity. Similarly, a Glu-259 in 257TLEE260 was selectively labeled using B. circulans β-galactosidase, indicating that Glu-259 is one of the nucleophiles in the active site. The present method can be readily extended to other glycosidases and should greatly aid the high throughput proteomics of many glycoside hydrolases showing both retaining- and inverting-type mechanisms.386Tuan, E.; Kirk, K. L. Fluorine reactivity in difluoromethylimidazoles. J. Fluorine Chem. 2006, 127, 980– 982, DOI: 10.1016/j.jfluchem.2006.03.014[Crossref], [CAS], Google Scholar386https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xmt1Gnu70%253D&md5=13ad34b686f5f888805ab05560782ef0Fluorine reactivity in difluoromethylimidazolesTuan, Edward; Kirk, Kenneth L.Journal of Fluorine Chemistry (2006), 127 (7), 980-982CODEN: JFLCAR; ISSN:0022-1139. (Elsevier B.V.)A difluoromethyl substituent attached directly to an imidazole ring is very reactive toward basic hydrolysis. A correlation of rate of fluoride loss with increasing pH is consistent with a mechanism that involves initial ionization of the imidazole NH, formation of an intermediate azafulvene by loss of HF, and reaction of the intermediate with solvent water.387Sakai, T. T.; Santi, D. V. Hydrolysis of hydroxybenzotrifluorides and fluorinated uracil derivatives. A general mechanism for carbon-fluorine bond labilization. J. Med. Chem. 1973, 16, 1079– 1084, DOI: 10.1021/jm00268a003[ACS Full Text], [CAS], Google Scholar387https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3sXlsVejsLc%253D&md5=c458d40df3311cf8e837efa35ab66f33Hydrolysis of hydroxybenzotrifluorides and fluorinated uracil derivatives. General mechanism for carbon-fluorine bond labilizationSakai, Ted T.; Santi, Daniel V.Journal of Medicinal Chemistry (1973), 16 (10), 1079-84CODEN: JMCMAR; ISSN:0022-2623.A general mechanism for C-F bond labilization was presented which may be predictively useful for study of enzyme mechanisms and in design of effective chemotherapeutic drugs. O-hydroxybenzotrifluoride (I) [444-30-4] and p-hydroxybenzotrifluoride [402-45-9] were hydrolyzed at alk. pH to the corresponding hydroxybenzoic acids, and the vinylog 1-trifluoromethyl-2-(4-hydroxyphenyl)ethylene [2338-84-3] was hydrolyzed to p-coumaric acid. 5-Difluoromethyluracil (II) [670-19-9] and 1-methyl-5-difluoromethyluracil [43064-01-3] were hydrolyzed to 5-formyluracil and 1-methyl-5-formyluracil, resp. Kinetics of hydrolysis of the phenolic fluoromethyl compds. suggested participation of the phenolate anions in displacement of fluoride ion to give difluoroquinone methide intermediates, e.g. III. Ability of the compds. to form olefinic intermediates appeared necessary for reaction to occur. A uracil anion probably participated in hydrolysis of the difluorouracil. Conversion of the olefinic intermediates to products presumably involved alternate addn. of nucleophile (or solvent) to the intermediate and elimination of F-.388Peukert, S.; Nunez, J.; He, F.; Dai, M.; Yusuff, N.; DiPesa, A.; Miller-Moslin, K.; Karki, R.; Lagu, B.; Harwell, C.; Zhang, Y.; Bauer, D.; Kelleher, J. F.; Egan, W. A method for estimating the risk of drug-induced phototoxicity and its application to smoothened inhibitors. MedChemComm 2011, 2, 973– 976, DOI: 10.1039/c1md00144b[Crossref], [CAS], Google Scholar388https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1Oqu7nK&md5=af0595f2f52ef672c7a42e32c6dab809A method for estimating the risk of drug-induced phototoxicity and its application to smoothened inhibitorsPeukert, Stefan; Nunez, Jill; He, Feng; Dai, Miao; Yusuff, Naeem; DiPesa, Alan; Miller-Moslin, Karen; Karki, Rajesh; Lagu, Bharat; Harwell, Chris; Zhang, Yalin; Bauer, Daniel; Kelleher, Joseph F.; Egan, WilliamMedChemComm (2011), 2 (10), 973-976CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)The energy difference between the frontier-orbital HOMO-LUMO gaps calcd. for the ground state of marketed oral drugs correlated with their obsd. phototoxicity. This mol. descriptor, together with their maximal molar absorptivity for UV light above 290 nm, can be used to predict phototoxicity risks. This is demonstrated for the phototoxicity mitigation of 1-piperazinyl phthalazines, a class of smoothened inhibitors.389Fournier, J. F.; Bouix-Peter, C.; Duvert, D.; Luzy, A. P.; Ouvry, G. Intrinsic property forecast index (iPFI) as a rule of thumb for medicinal chemists to remove a phototoxicity liability. J. Med. Chem. 2018, 61, 3231– 3236, DOI: 10.1021/acs.jmedchem.8b00075[ACS Full Text], [CAS], Google Scholar389https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXltVGksb8%253D&md5=d9a034f7ce81e84d099e94d26a0573e5Intrinsic Property Forecast Index (iPFI) as a Rule of Thumb for Medicinal Chemists to Remove a Phototoxicity LiabilityFournier, Jean-Francois; Bouix-Peter, Claire; Duvert, Denis; Luzy, Anne-Pascale; Ouvry, GillesJournal of Medicinal Chemistry (2018), 61 (7), 3231-3236CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Phototoxicity occurs when UV irradn. causes otherwise benign compds. to become irritant, sensitizers, or even genotoxic. This toxicity is particularly a concern after topical application and in dermatol. programs where skin irritation can be incompatible with the desired therapeutic outcome. This brief article establishes that the intrinsic property forecast index (iPFI) can be used to evaluate the probability of a compd. being phototoxic and gives medicinal chemists a practical tool to handle this liability.390Schmidt, F.; Wenzel, J.; Halland, N.; Gussregen, S.; Delafoy, L.; Czich, A. Computational investigation of drug phototoxicity: photosafety assessment, photo-toxophore identification, and machine learning. Chem. Res. Toxicol. 2019, 32, 2338– 2352, DOI: 10.1021/acs.chemrestox.9b00338[ACS Full Text], [CAS], Google Scholar390https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvFOrurvP&md5=c181094b301a4c59ef3ac546aada70e0Computational Investigation of Drug Phototoxicity: Photosafety Assessment, Photo-Toxophore Identification, and Machine LearningSchmidt, Friedemann; Wenzel, Jan; Halland, Nis; Guessregen, Stefan; Delafoy, Laure; Czich, AndreasChemical Research in Toxicology (2019), 32 (11), 2338-2352CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)One of the most appreciated capabilities of computational toxicol. is to support the design of pharmaceuticals with reduced toxicol. hazard. To this end, we have strengthened our drug photosafety assessments by applying novel computer models for the anticipation of in vitro phototoxicity and human photosensitization. These models are typically used in pharmaceutical discovery projects as part of the compd. toxicity assessments and compd. optimization methods. To ensure good data quality and aiming at models with global applicability we sep. compiled and curated highly chem. diverse data sets from 3T3 NRU phototoxicity reports (450 compds.) and clin. photosensitization alerts (1419 compds.) which are provided as supplements. The latter data gives rise to a comprehensive list of explanatory fragments for visual guidance, termed phototoxophores, by application of a Bayesian statistics approach. To extend beyond the domain of well sampled fragments we applied machine learning techniques based on explanatory descriptors such as pharmacophoric fingerprints or, more important, accurate electronic energy descriptors. Electronic descriptors were extd. from quantum chem. computations at the d. functional theory (DFT) level. Accurate UV/vis spectral absorption descriptors and pharmacophoric fingerprints turned out to be necessary for predictive computer models, which were both derived from Deep Neural Networks but also the simpler Random Decision Forests approach. Model accuracies of 83-85% could typically be reached for diverse test data sets and other company inhouse data, while model sensitivity (the capability of correctly detecting toxicants) was even better, reaching 86%-90%. Importantly, a computer model-triggered response-map allowed for graphical/chem. interpretability also in the case of previously unknown phototoxophores. The photosafety models described here are currently applied in a prospective manner for the hazard identification, prioritization, and optimization of newly designed mols.391Miolo, G.; Levorato, L.; Gallocchio, F.; Caffieri, S.; Bastianon, C.; Zanoni, R.; Reddi, E. In vitro phototoxicity of phenothiazines: involvement of stable UVA photolysis products formed in aqueous medium. Chem. Res. Toxicol. 2006, 19, 156– 163, DOI: 10.1021/tx0502239[ACS Full Text], [CAS], Google Scholar391https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlWmu7zP&md5=d7376f883194fac567f40cf231d6d34dIn Vitro Phototoxicity of Phenothiazines: Involvement of Stable UVA Photolysis Products Formed in Aqueous MediumMiolo, G.; Levorato, L.; Gallocchio, F.; Caffieri, S.; Bastianon, C.; Zanoni, R.; Reddi, E.Chemical Research in Toxicology (2006), 19 (1), 156-163CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)This paper reports the results of an in vitro evaluation of the phototoxic potential of stable photoproducts formed by UVA photolysis of three phenothiazines, perphenazine, fluphenazine, and thioridazine, in a water environment. Perphenazine gave a single product due to dechlorination. From thioridazine, the two major products formed; the endocyclic sulfoxide and the endocyclic N-oxide in which the 2-SCH3 substituent was replaced by a hydroxy group were tested. From fluphenazine, two products have been examd. as follows: an exocyclic N-piperazine oxide and a carboxylic acid arising from hydrolysis of the 2-CF3 group. The phototoxicity of the isolated photoproducts has been studied to det. their possible involvement in the photosensitizing effects exhibited by the parent drugs, using hemolysis and 3T3 fibroblasts viability as in vitro assays. As fluphenazine, perphenazine, and thioridazine did, some photoproducts proved phototoxic. In particular, the perphenazine dechlorinated photoproduct and the thioridazine N-oxide were found to exert phototoxic properties similar to the parent compds. Therefore, our data suggest that some phenothiazine photoproducts may play a role in the mechanism of photosensitivity of these drugs. Because some of these photoproducts correspond to metabolic products of phenothiazines found in humans, it cannot be ruled out that metabolites of phenothiazines can be phototoxic in vivo.392Caffieri, S.; Miolo, G.; Seraglia, R.; Dalzoppo, D.; Toma, F. M.; Beyersbergen van Henegouwen, G. M. J. Photoaddition of fluphenazine to nucleophiles in peptides and proteins. Possible cause of immune side effects. Chem. Res. Toxicol. 2007, 20, 1470– 1476, DOI: 10.1021/tx700123u[ACS Full Text], [CAS], Google Scholar392https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVKrs73P&md5=1cb0a2a1f2df32520d1c3cf4416bfaf9Photoaddition of Fluphenazine to Nucleophiles in Peptides and Proteins. Possible Cause of Immune Side EffectsCaffieri, Sergio; Miolo, Giorgia; Seraglia, Roberta; Dalzoppo, Daniele; Toma, Francesca M.; Beyersbergen van Henegouwen, Gerard M. J.Chemical Research in Toxicology (2007), 20 (10), 1470-1476CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)By the action of UVA light, fluphenazine reacted with nucleophiles through a mechanism involving defluorination of its trifluoromethyl group, giving rise to carboxylic acid derivs. that were easily detected by electrospray mass spectrometry. This photoreaction took place with alcs., sulphydryls, and amines. When irradn. of fluphenazine was carried out in the presence of an amino acid at pH 7.4, the α-amino group was covalently bound to the drug. With amino acids possessing a further nucleophilic residue on the side chain, such as lysine, tyrosine, and cysteine-but not serine-both groups reacted, resulting in a fluphenazine-amino acid-fluphenazine diadduct. The same occurred with the physiol. peptide glutathione (γ-glutamylcysteinylglycine). By means of MALDI mass spectrometry, it was shown that fluphenazine also covalently bound to peptides and proteins such as calmodulin. This binding may result in the formation of antibodies, ultimately leading to the destruction of the granulocytes and thus suggesting that photoactivation of this drug may play a role in its clin. side effects, such as agranulocytosis.393Lam, M. W.; Young, C. J.; Mabury, S. A. Aqueous photochemical reaction kinetics and transformations of fluoxetine. Environ. Sci. Technol. 2005, 39, 513– 522, DOI: 10.1021/es0494757[ACS Full Text], [CAS], Google Scholar393https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtVCrsbvJ&md5=522617f27e634f4a4a7b80806b1d2d34Aqueous Photochemical Reaction Kinetics and Transformations of FluoxetineLam, Monica W.; Young, Cora J.; Mabury, Scott A.Environmental Science and Technology (2005), 39 (2), 513-522CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)Fluoxetine (FLX) was shown to be photoreactive in sunlit surface water. FLX degraded in deionized water when exposed to simulated sunlight with a half-life of 55.2 ± 3.6 h. Photodegrdn. products were identified by HPLC-UV and liq. chromatog.-tandem mass spectrometry (LC-MS-MS) with electro-spray (ES) ionization. Defluorination of the trifluoromethyl group in FLX and in fluometuron and flutalanil, 2 other compds. contg. this functional group, was suggested to be a common direct photolysis pathway for trifluoromethylated compds.; products resulting from O-dealkylation of FLX were also obsd. The degrdn. rate was faster in synthetic field water where OH- was the likely dominant system oxidant. The bimol. rate const. for the reaction between FLX and OH- was measured as 8.4 ± 0.5 × 109 and 9.6 ± 0.8 × 109 M/s using 2 different methods of competition kinetics. Indirect photodegrdn. reactions could lead to prodn. of hydroxylated and O-dealkylated compds. Although direct photolysis could potentially limit the persistence of FLX in surface water, its degrdn. by indirect photolysis would proceed faster. Thus, the latter process could be important in eliminating FLX in surface water.394Bosca, F.; Cuquerella, M. C.; Marin, M. L.; Miranda, M. A. Photochemistry of 2-hydroxy-4-trifluoromethylbenzoic acid, major metabolite of the photosensitizing platelet antiaggregant drug triflusal. Photochem. Photobiol. 2001, 73, 463– 468, DOI: 10.1562/0031-8655(2001)0730463POHTAM2.0.CO2[Crossref], [PubMed], [CAS], Google Scholar394https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjsFCkt7Y%253D&md5=d540822aa7602ca2767adac042743f08Photochemistry of 2-hydroxy-4-trifluoromethylbenzoic acid, major metabolite of the photosensitizing platelet antiaggregant drug triflusalBosca, F.; Cuquerella, M. C.; Marin, M. L.; Miranda, M. A.Photochemistry and Photobiology (2001), 73 (5), 463-468CODEN: PHCBAP; ISSN:0031-8655. (American Society for Photobiology)Triflusal is a platelet antiaggregant drug with photoallergic side effects. However, it is considered a prodrug since it is metabolized to 2-hydroxy-4-trifluoromethyl-benzoic acid (HTB)-the pharmacol. active form. HTB was found to be photolabile under various conditions. Its major photodegrdn. pathway appears to be the nucleophilic attack at the trifluoromethyl moiety. The involvement of the triplet state in the photodegrdn. has been unequivocally proved by direct detection of this transient in laser flash photolysis and by quenching expts. with oxygen, cyclohexadiene and naphthalene. Finally, the photobinding of HTB to proteins such as bovine serum albumin has been demonstrated using UV-visible (UV-Vis) and fluorescence spectroscopy. Nucleophilic groups present in the protein appear to be responsible for the formation of covalent drug photoadducts, which is the first step involved in the photoallergy shown by triflusal.395Nuin, E.; Perez-Sala, D.; Lhiaubet-Vallet, V.; Andreu, I.; Miranda, M. A. Photosensitivity to triflusal: formation of a photoadduct with ubiquitin demonstrated by photophysical and proteomic techniques. Front. Pharmacol. 2016, 7, 277, DOI: 10.3389/fphar.2016.00277[Crossref], [PubMed], [CAS], Google Scholar395https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFSisrbO&md5=8d2999547cef362049c5dece94c8b49aPhotosensitivity to triflusal: formation of a photoadduct with ubiquitin demonstrated by photophysical and proteomic techniquesNuin, Edurne; Perez-Sala, Dolores; Lhiaubet-Vallet, Virginie; Andreu, Inmaculada; Miranda, Miguel A.Frontiers in Pharmacology (2016), 7 (), 277/1-277/8CODEN: FPRHAU; ISSN:1663-9812. (Frontiers Media S.A.)Triflusal is a platelet aggregation inhibitor chem. related to acetylsalicylic acid, which is used for the prevention and/or treatment of vascular thromboembolisms, which acts as a prodrug. Actually, after oral administration it is absorbed primarily in the small intestine, binds to plasma proteins (99%) and is rapidly biotransformed in the liver into its deacetylated active metabolite 2-hydroxy-4-trifluoromethylbenzoic acid (HTB). In healthy humans, the half-life of triflusal is ca. 0.5 h, whereas for HTB it is ca. 35 h. From a pharmacol. point of view, it is interesting to note that HTB is itself highly active as a platelet anti-aggregant agent. Indeed, studies on the clin. profile of both drug and metabolite have shown no significant differences between them. It has been evidenced that HTB displays ability to induce photoallergy in humans. This phenomenon involves a cell-mediated immune response, which is initiated by covalent binding of a light-activated photosensitizer (or a species derived therefrom) to a protein. In this context, small proteins like ubiquitin could be appropriate models for investigating covalent binding by means of MS/MS and peptide fingerprint anal. In previous work, it was shown that HTB forms covalent photoadducts with isolated lysine. Interestingly, ubiquitin contains seven lysine residues that could be modified by a similar reaction. With this background, the aim of the present work is to explore adduct formation between the triflusal metabolite and ubiquitin as model protein upon sunlight irradn., combining proteomic and photophys. (fluorescence and laser flash photolysis) techniques. Photophys. and proteomic anal. demonstrates monoadduct formation as the major outcome of the reaction. Interestingly, addn. can take place at any of the ε-amino groups of the lysine residues of the protein and involves replacement of the trifluoromethyl moiety with a new amide function. This process can in principle occur with other trifluoroarom. compds. and may be responsible for the appearance of undesired photoallergic side effects.396Chaignon, P.; Cortial, S.; Guerineau, V.; Adeline, M. T.; Giannotti, C.; Fan, G.; Ouazzani, J. Photochemical reactivity of trifluoromethyl aromatic amines: the example of 3,5-diamino-trifluoromethyl-benzene (3,5-DABTF). Photochem. Photobiol. 2005, 81, 1539– 1543, DOI: 10.1562/2005-08-03-RA-637[Crossref], [PubMed], [CAS], Google Scholar396https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlekt7%252FF&md5=ac29f22dfd1188e91fd0dc2dec99bd3cPhotochemical reactivity of trifluoromethyl aromatic amines: The example of 3,5-diamino-trifluoromethyl-benzene (3,5-DABTF)Chaignon, Philippe; Cortial, Sylvie; Guerineau, Vincent; Adeline, Marie-Therese; Giannotti, Charles; Fan, Gerard; Ouazzani, JamalPhotochemistry and Photobiology (2005), 81 (Nov./Dec.), 1539-1543CODEN: PHCBAP; ISSN:0031-8655. (American Society for Photobiology)This work presents the application of an online photoreactor to the detection of 3,5-diamino-trifluoromethyl-benzene (3,5-DABTF) in aq. solns. When irradiated at 310 nm, this compd. is defluorinated to 3,5-diaminobenzoic acid by a nucleophilic substitution of the fluoride by water. Concomitantly, defluorination intermediates polymerize through amide bonds to give dark-colored compds. We take advantage of the photocatalyzed defluorination and the subsequent decrease in pH to develop an original and specific photoreactor. Continuous recording of pH and temp. in the outlet of the reactor by a dual electrode gives us an opportunity to optimize the system. In the photoreactor, 3,5-DABTF is immediately and totally transformed as attested by the rapid drop of the flowing soln. pH from 6.2 to 3.2 and the chromatog. anal. of the outgoing solns. The detection remains effective from 1 to 1000 ppm.397Perez-Ruiz, R.; Molins-Molina, O.; Lence, E.; Gonzalez-Bello, C.; Miranda, M. A.; Jimenez, M. C. Photogeneration of quinone methides as latent electrophiles for lysine targeting. J. Org. Chem. 2018, 83, 13019– 13029, DOI: 10.1021/acs.joc.8b01559[ACS Full Text], [CAS], Google Scholar397https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvVaqtL7O&md5=c8c0eca3cbea34b02c18dbc55d40464cPhotogeneration of Quinone Methides as Latent Electrophiles for Lysine TargetingPerez-Ruiz, Raul; Molins-Molina, Oscar; Lence, Emilio; Gonzalez-Bello, Concepcion; Miranda, Miguel A.; Jimenez, M. ConsueloJournal of Organic Chemistry (2018), 83 (21), 13019-13029CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)Latent electrophiles are nowadays very attractive chem. entities for drug discovery, as they are unreactive unless activated upon binding with the specific target. In this work, the utility of 4-trifluoromethyl phenols as precursors of latent electrophiles, quinone methides (QM), for lysine-targeting is demonstrated. These Michael acceptors were photogenerated for specific covalent modification of lysine residues using human serum albumin (HSA) as a model target. The reactive QM-type intermediates I or II, generated upon irradn. of 4-trifluoromethyl-1-naphthol (1)@HSA or 4-(4-trifluoromethylphenyl)phenol (2)@HSA complexes, exhibited chemoselective reactivity toward lysine residues leading to amide adducts, which was confirmed by proteomic anal. For ligand 1, the covalent modification of residues Lys106 and Lys414 (located in subdomains IA and IIIA, resp.) was obsd., whereas for ligand 2, the modification of Lys195 (in subdomain IIA) took place. Docking and mol. dynamics simulation studies provided an insight into the mol. basis of the selectivity of 1 and 2 for these HSA subdomains and the covalent modification mechanism. These studies open the opportunity of performing protein silencing by generating reactive ligands under very mild conditions (irradn.) for specific covalent modification of hidden lysine residues.398Lanfranchi, D. A.; Belorgey, D.; Muller, T.; Vezin, H.; Lanzer, M.; Davioud-Charvet, E. Exploring the trifluoromenadione core as a template to design antimalarial redox-active agents interacting with glutathione reductase. Org. Biomol. Chem. 2012, 10, 4795– 4806, DOI: 10.1039/c2ob25229e[Crossref], [PubMed], [CAS], Google Scholar398https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XnvVSrt7w%253D&md5=1f8a8106ab91264fdec3f2e1ab85b769Exploring the trifluoromenadione core as a template to design antimalarial redox-active agents interacting with glutathione reductaseLanfranchi, Don Antoine; Belorgey, Didier; Mueller, Tobias; Vezin, Herve; Lanzer, Michael; Davioud-Charvet, ElisabethOrganic & Biomolecular Chemistry (2012), 10 (24), 4795-4806CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)Menadione is the 2-methyl-1,4-naphthoquinone core used to design potent antimalarial redox-cyclers to affect the redox equil. of Plasmodium-infected red blood cells. Exploring the reactivity of fluoromethyl-1,4-naphthoquinones, in particular trifluoromenadione, under quasi-physiol. conditions in NADPH-dependent glutathione reductase reactions, is discussed in terms of chem. synthesis, electrochem., enzyme kinetics, and antimalarial activities. Multitarget-directed drug discovery is an emerging approach to the design of new antimalarial drugs. Combining in one single 1,4-naphthoquinone mol., the trifluoromenadione core with the alkyl chain at C-3 of the known antimalarial drug atovaquone, revealed a mechanism for CF3 as a leaving group. The resulting trifluoromethyl deriv. 5 showed a potent antimalarial activity per se against malarial parasites in culture.399Johann, L.; Belorgey, D.; Huang, H. H.; Day, L.; Chesse, M.; Becker, K.; Williams, D. L.; Davioud-Charvet, E. Synthesis and evaluation of 1,4-naphthoquinone ether derivatives as SmTGR inhibitors and new anti-schistosomal drugs. FEBS J. 2015, 282, 3199– 3217, DOI: 10.1111/febs.13359[Crossref], [PubMed], [CAS], Google Scholar399https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1KmurjK&md5=99d3b64dd06ee4442ea4c9a916417931Synthesis and evaluation of 1,4-naphthoquinone ether derivatives as SmTGR inhibitors and new anti-schistosomal drugsJohann, Laure; Belorgey, Didier; Huang, Hsin-Hung; Day, Latasha; Chesse, Matthieu; Becker, Katja; Williams, David L.; Davioud-Charvet, ElisabethFEBS Journal (2015), 282 (16), 3199-3217CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)New 3-benzyloxomenadiones and their difluoromethylmenadione derivs. I (R1 = Me, CHF2; R2 = H, F; R3 = COOH, CN, Cl, H, Br, OMe; R4 = CF3) were synthesized and were found to be potent and specific inhibitors of Schistosoma mansoni thioredoxin-glutathione reductase (SmTGR), which has been identified as a potential target for anti-schistosomal drugs. The compds. were also tested in enzymic assays using both human flavoenzymes, i.e. glutathione reductase (hGR) and selenium-dependent human thioredoxin reductase (hTrxR), to evaluate the specificity of the inhibition. Structure-activity relationships as well as physico- and electro-chem. studies showed a high potential for the 3-phenoxymethyl menadiones to inhibit SmTGR selectively compared to hGR and hTrxR enzymes, in particular those bearing an α-fluorophenol Me ether moiety, which improves anti-schistosomal action. Furthermore, the (substituted phenoxy)methyl menadione deriv. I (R1 = Me; R2 = H; R3 = OMe; R4 = CF3) displayed time-dependent SmTGR inactivation, correlating with unproductive NADPH-dependent redox cycling of SmTGR, and potent anti-schistosomal action in worms cultured ex vivo. In contrast, the difluoromethylmenadione analog I (R1 = CHF2; R2 = H; R3 = OMe; R4 = CF3), which inactivates SmTGR through an irreversible non-consuming NADPH-dependent process, has little killing effect in worms cultured ex vivo. Despite ex vivo activity, none of the compds. tested was active in vivo.400Eckstein, J. W.; Foster, P. G.; Finer-Moore, J.; Wataya, Y.; Santi, D. V. Mechanism-based inhibition of thymidylate synthase by 5-(trifluoromethyl)-2′-deoxyuridine 5′-monophosphate. Biochemistry 1994, 33, 15086– 15094, DOI: 10.1021/bi00254a018[ACS Full Text], [CAS], Google Scholar400https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXmvFGrt70%253D&md5=f82c290ac5cb6d16115a3517f283477aMechanism-Based Inhibition of Thymidylate Synthase by 5-(Trifluoromethyl)-2'-deoxyuridine 5'-MonophosphateEckstein, Jens W.; Foster, Paul G.; Finer-Moore, Janet; Wataya, Yusuke; Santi, Daniel V.Biochemistry (1994), 33 (50), 15086-94CODEN: BICHAW; ISSN:0006-2960.Thymidylate synthase (TS) from Lactobacillus casei is inhibited by 5-(trifluoromethyl)-2'-deoxyuridine 5'-monophosphate (CF3dUMP). CF3dUMP binds to the active site of TS in the absence of CH2H4folate, and attack of the catalytic nucleophile cysteine 198 at C6 of the pyrimidine leads to activation of the trifluoromethyl group and release of fluoride ion. Subsequently, the activated heterocycle reacts with a nucleophile of the enzyme to form a moderately stable covalent complex. Proteolytic digestion of TS treated with [2'-3H]CF3dUMP, followed by sequencing of the labeled peptides, revealed that tyrosine 146 and cysteine 198 are covalently bound to the inhibitor in the enzyme-inhibitor complex. The presence of dithiothreitol (DTT) or β-mercaptoethanol resulted in the breakdown of the covalent complex, and products from the breakdown of the complex were isolated and characterized. The three-dimensional structure of the enzyme-inhibitor complex was detd. by x-ray crystallog., clearly demonstrating covalent attachment of the nucleotide to tyrosine 146. A chem. reaction mechanism for the inhibition of TS by CF3dUMP is presented that is consistent with the kinetic, biochem., and structural results.401Begue, J.-P.; Bonnet-Delpon, D. Recent advances (1995–2005) in fluorinated pharmaceuticals based on natural products. J. Fluorine Chem. 2006, 127, 992– 1012, DOI: 10.1016/j.jfluchem.2006.05.006[Crossref], [CAS], Google Scholar401https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XnvVSru78%253D&md5=4741870ef64899a173607fecb4cf66beRecent advances (1995-2005) in fluorinated pharmaceuticals based on natural productsBegue, Jean-Pierre; Bonnet-Delpon, DanieleJournal of Fluorine Chemistry (2006), 127 (8), 992-1012CODEN: JFLCAR; ISSN:0022-1139. (Elsevier B.V.)A review. This present report is devoted to the recent advances, in these last 10 years, in fluorinated analogs of natural products developed as pharmaceuticals, marketed, registered or in clin. development. These mainly concern fluorine-substituted nucleosides, alkaloids, macrolides, steroids, amino acids and prostaglandins.402Santi, D. V.; Sakai, T. T. Thymidylate synthetase. Model studies of inhibition by 5-trifluoromethyl-2’-deoxyuridylic acid. Biochemistry 1971, 10, 3598– 3607, DOI: 10.1021/bi00795a018[ACS Full Text], [CAS], Google Scholar402https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE38XhtF2l&md5=c2006c61dbce0f15ec1a03f02f671d4eThymidylate synthetase. Model studies of inhibition by 5-trifluoromethyl-2'-deoxyuridylic acidSanti, Daniel V.; Sakai, Ted T.Biochemistry (1971), 10 (19), 3598-607CODEN: BICHAW; ISSN:0006-2960.The mechanisms of hydrolytic reactions of 5-trifluoromethyluracil and its N-alkylated derivs. provide insight into the mechanism of irreversible inhibition of thymidylate synthetase by 5-trifluoromethyl-2'-deoxyuridylic acid. All reactions appear to proceed by formation of a highly reactive intermediate having an exocyclic difluoromethylene group at the 5 position which subsequently reacts with water or hydroxide ion in a series of rapid steps to give corresponding 5-carboxyuracils. An analogous mechanism for the acylation of thymidylate synthetase by 5-trifluoromethyl-2'-deoxyuridine is proposed in which a nucleophilic group of the enzyme active-site participates in the activation of the trifluoromethyl group.403Pettersson, M.; Hou, X.; Kuhn, M.; Wager, T. T.; Kauffman, G. W.; Verhoest, P. R. Quantitative assessment of the impact of fluorine substitution on P-glycoprotein (P-gp) mediated efflux, permeability, lipophilicity, and metabolic stability. J. Med. Chem. 2016, 59, 5284– 5296, DOI: 10.1021/acs.jmedchem.6b00027[ACS Full Text], [CAS], Google Scholar403https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xos1Kjsro%253D&md5=7ca62f3c1100c6623d082d88b3113cf8Quantitative Assessment of the Impact of Fluorine Substitution on P-Glycoprotein (P-gp) Mediated Efflux, Permeability, Lipophilicity, and Metabolic StabilityPettersson, Martin; Hou, Xinjun; Kuhn, Max; Wager, Travis T.; Kauffman, Gregory W.; Verhoest, Patrick R.Journal of Medicinal Chemistry (2016), 59 (11), 5284-5296CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Strategic replacement of one or more hydrogen atoms with fluorine atom(s) is a common tactic to improve potency at a given target and/or to modulate parameters such as metabolic stability and pKa. Mol. wt. (MW) is a key parameter in design, and incorporation of fluorine is assocd. with a disproportionate increase in MW considering the van der Waals radius of fluorine vs. hydrogen. Herein we examine a large compd. data set to understand the effect of introducing fluorine on the risk of encountering P-glycoprotein mediated efflux (as measured by MDR efflux ratio), passive permeability, lipophilicity, and metabolic stability. Statistical modeling of the MDR ER data demonstrated that an increase in MW as a result of introducing fluorine atoms does not lead to higher risk of P-gp mediated efflux. Fluorine-cor. mol. wt. (MWFC), where the mol. wt. of fluorine has been subtracted, was found to be a more relevant descriptor.404Tan, E. Y.; Hartmann, G.; Chen, Q.; Pereira, A.; Bradley, S.; Doss, G.; Zhang, A. S.; Ho, J. Z.; Braun, M. P.; Dean, D. C.; Tang, W.; Kumar, S. Pharmacokinetics, metabolism, and excretion of anacetrapib, a novel inhibitor of the cholesteryl ester transfer protein, in rats and rhesus monkeys. Drug Metab. Dispos. 2010, 38, 459– 473, DOI: 10.1124/dmd.109.028696[Crossref], [PubMed], [CAS], Google Scholar404https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXjt1OrtL4%253D&md5=fe04465bd0c53e02e295698e0e038f6aPharmacokinetics, metabolism, and excretion of anacetrapib, a novel inhibitor of the cholesteryl ester transfer protein, in rats and rhesus monkeysTan, Eugene Y.; Hartmann, Georgy; Chen, Qing; Pereira, Antonio; Bradley, Scott; Doss, George; Zhang, Andy Shiqiang; Ho, Jonathan Z.; Braun, Matthew P.; Dean, Dennis C.; Tang, Wei; Kumar, SanjeevDrug Metabolism and Disposition (2010), 38 (3), 459-473CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)The pharmacokinetics and metab. of anacetrapib (MK-0859), a novel cholesteryl ester transfer protein inhibitor, were examd. in rats and rhesus monkeys. Anacetrapib exhibited a low clearance in both species and a moderate oral bioavailability of ∼38% in rats and ∼13% in monkeys. The area under the plasma concn.-time curve in both species increased in a less than dose-proportional manner over an oral dose range of 1 to 500 mg/kg. After oral administration of [14C]anacetrapib at 10 mg/kg, ∼80 and 90% of the radioactive dose was recovered over 48 h postdose from rats and monkeys, resp. The majority of the administered radioactive dose was excreted unchanged in feces in both species. Biliary excretion of radioactivity accounted for ∼15% and urinary excretion for less than 2% of the dose. Thirteen metabolites, resulting from oxidative and secondary glucuronic acid conjugation, were identified in rat and monkey bile. The main metabolic pathways consisted of O-demethylation (M1) and hydroxylation on the biphenyl moiety (M2) and hydroxylation on the iso-Pr side chain (M3); these hydroxylations were followed by O-glucuronidation of these metabolites. A glutathione adduct (M9), an olefin metabolite (M10), and a propionic acid metabolite (M11) also were identified. In addn. to parent anacetrapib, M1, M2, and M3 metabolites were detected in rat but not in monkey plasma. Overall, it appears that anacetrapib exhibits a low-to-moderate degree of absorption after oral dosing and majority of the absorbed dose is eliminated via oxidn. to a series of hydroxylated metabolites that undergo conjugation with glucuronic acid before excretion into bile.405Bowman, L.; Hopewell, J. C.; Chen, F.; Wallendszus, K.; Stevens, W.; Collins, R.; Wiviott, S. D.; Cannon, C. P.; Braunwald, E.; Sammons, E.; Landray, M. J. Effects of anacetrapib in patients with atherosclerotic vascular disease. N. Engl. J. Med. 2017, 377, 1217– 1227, DOI: 10.1056/NEJMoa1706444[Crossref], [PubMed], [CAS], Google Scholar405https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1cbit1Gmsg%253D%253D&md5=8f152d6a2e2d83df68f4b511bf59164bEffects of Anacetrapib in Patients with Atherosclerotic Vascular DiseaseBowman Louise; Hopewell Jemma C; Chen Fang; Wallendszus Karl; Stevens William; Collins Rory; Wiviott Stephen D; Cannon Christopher P; Braunwald Eugene; Wiviott Stephen D; Cannon Christopher P; Braunwald Eugene; Sammons Emily; Landray Martin JThe New England journal of medicine (2017), 377 (13), 1217-1227 ISSN:.BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .).406Gotto, A. M., Jr.; Cannon, C. P.; Li, X. S.; Vaidya, S.; Kher, U.; Brinton, E. A.; Davidson, M.; Moon, J. E.; Shah, S.; Dansky, H. M.; Mitchel, Y.; Barter, P. Evaluation of lipids, drug concentration, and safety parameters following cessation of treatment with the cholesteryl ester transfer protein inhibitor anacetrapib in patients with or at high risk for coronary heart disease. Am. J. Cardiol. 2014, 113, 76– 83, DOI: 10.1016/j.amjcard.2013.08.041[Crossref], [PubMed], [CAS], Google Scholar406https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhslajsLzN&md5=fa70e20a5a463a78c9df31c4bf66f588Evaluation of Lipids, Drug Concentration, and Safety Parameters Following Cessation of Treatment With the Cholesteryl Ester Transfer Protein Inhibitor Anacetrapib in Patients With or at High Risk for Coronary Heart DiseaseGotto, Antonio M.; Cannon, Christopher P.; Li, Xiujiang Susie; Vaidya, Sanskruti; Kher, Uma; Brinton, Eliot A.; Davidson, Michael; Moon, Jennifer E.; Shah, Sukrut; Dansky, Hayes M.; Mitchel, Yale; Barter, PhilipAmerican Journal of Cardiology (2014), 113 (1), 76-83CODEN: AJCDAG; ISSN:0002-9149. (Elsevier)The aim of this study was to assess the effects on lipids and safety during a 12-wk reversal period after 18 mo of treatment with anacetrapib. The cholesteryl ester transfer protein inhibitor anacetrapib was previously shown to reduce low-d. lipoprotein cholesterol by 39.8% (estd. using the Friedewald equation) and increase high-d. lipoprotein (HDL) cholesterol by 138.1%, with an acceptable side-effect profile, in patients with or at high risk for coronary heart disease in the Detg. the Efficacy and Tolerability of CETP Inhibition With Anacetrapib (DEFINE) trial. A total of 1,398 patients entered the 12-wk reversal-phase study, either after completion of the active-treatment phase or after early discontinuation of the study medication. In patients allocated to anacetrapib, placebo-adjusted mean percentage decreases from baseline were obsd. at 12 wk off the study drug for Friedewald-calcd. low-d. lipoprotein cholesterol (18.6%), non-HDL cholesterol (17.6%), and apolipoprotein B (10.2%); placebo-adjusted mean percentage increases were obsd. for HDL cholesterol (73.0%) and apolipoprotein A-I (24.5%). Residual plasma anacetrapib levels (about 40% of on-treatment apparent steady-state trough levels) were also detected 12 wk after cessation of anacetrapib. No clin. important elevations in liver enzymes, blood pressure, electrolytes, or adverse experiences were obsd. during the reversal phase. Preliminary data from a small cohort (n = 30) revealed the presence of low concns. of anacetrapib in plasma 2.5 to 4 years after the last anacetrapib dose. In conclusion, after the cessation of active treatment, anacetrapib plasma lipid changes and drug levels decreased to approx. 40% of on-treatment trough levels at 12 wk after dosing, but modest HDL cholesterol elevations and low drug concns. were still detectable 2 to 4 years after the last dosing.407Johns, D. G.; LeVoci, L.; Krsmanovic, M.; Lu, M.; Hartmann, G.; Xu, S.; Wang, S. P.; Chen, Y.; Bateman, T.; Blaustein, R. O. Characterization of anacetrapib distribution into the lipid droplet of adipose tissue in mice and human cultured adipocytes. Drug Metab. Dispos. 2019, 47, 227– 233, DOI: 10.1124/dmd.118.084525[Crossref], [PubMed], [CAS], Google Scholar407https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXotlKiu7k%253D&md5=b8437c0827e69d455fa217148e288fc6Characterization of anacetrapib distribution into the lipid droplet of adipose tissue in mice and human cultured adipocytesJohns, Douglas G.; Levoci, Lauretta; Krsmanovic, Mihajlo; Lu, Min; Hartmann, Georgy; Xu, Suoyu; Wang, Sheng-Ping; Chen, Ying; Bateman, Thomas; Blaustein, Robert O.Drug Metabolism & Disposition (2019), 47 (3), 227-233CODEN: DMDSAI; ISSN:1521-009X. (American Society for Pharmacology and Experimental Therapeutics)Anacetrapib is an inhibitor of cholesteryl ester transfer protein (CETP), assocd. with redn. in LDL cholesterol and increase in HDL cholesterol in hypercholesterolemic patients. Anacetrapib was not taken forward into filing/registration as a newdrug for coronary artery diease, despite the observation of a ∼9% redn. in cardiovascular risk in a large phase III cardiovascular outcomes trial (REVEAL). Anacetrapib displayed no adverse effects throughout extensive preclin. safety evaluation, and no major safety signals were obsd. in clin. trials studying anacetrapib, including REVEAL. However, anacetrapib demonstrated a long terminal half-life in all species, thought to be due, in part, to distribution into adipose tissue. We sought to understand the dependence of anacetrapib's long half-life on adipose tissue and to explore potential mechanisms that might contribute to the phenomenon. In mice, anacetrapib localized primarily to the lipid droplet of adipocytes in white adipose tissue; in vitro, anacetrapib entry into cultured human adipocytes depended on the presence of a mature adipocyte and lipid droplet but did not require active transport. In vivo, the entry of anacetrapib into adipose tissue did not require lipase activity, as the distribution of anacetrapib into adipose was-not affected by systemic lipase inhibition using poloaxamer-407, a systemic lipase inhibitor. The data from these studies support the notion that the entry of anacetrapib into adipose tissue/lipid droplets does not require active transport, nor does it require mobilization or entry of fat into adipose via lipolysis.408Hartmann, G.; Kumar, S.; Johns, D.; Gheyas, F.; Gutstein, D.; Shen, X.; Burton, A.; Lederman, H.; Lutz, R.; Jackson, T.; Chavez-Eng, C.; Mitra, K. Disposition into adipose tissue determines accumulation and elimination kinetics of the cholesteryl ester transfer protein inhibitor anacetrapib in mice. Drug Metab. Dispos. 2016, 44, 428– 434, DOI: 10.1124/dmd.115.067736[Crossref], [PubMed], [CAS], Google Scholar408https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28rlsVSlsw%253D%253D&md5=889e0108f486863be71af1807c6e4f40Disposition into Adipose Tissue Determines Accumulation and Elimination Kinetics of the Cholesteryl Ester Transfer Protein Inhibitor Anacetrapib in MiceHartmann Georgy; Kumar Sanjeev; Johns Douglas; Gheyas Ferdous; Gutstein David; Shen Xiaolan; Burton Aimee; Lederman Harmony; Lutz Ryan; Jackson Tonya; Chavez-Eng Cynthia; Mitra KaushikDrug metabolism and disposition: the biological fate of chemicals (2016), 44 (3), 428-34 ISSN:.The cholesteryl ester transfer protein (CETP) inhibitor anacetrapib exhibits a long terminal half-life (t1/2) in humans; however, the dispositional mechanisms that lead to this long t1/2 are still being elucidated. As it is hypothesized that disposition into adipose tissue and binding to CETP might play a role, we sought to delineate the relative importance of these factors using a preclinical animal model. A multiple-dose pharmacokinetic study was conducted in C57BL6 wild-type (WT) lean, WT diet-induced obese (DIO), natural flanking region (NFR) CETP-transgenic lean, and NFR-DIO mice. Mice were dosed orally with 10 mg/kg anacetrapib daily for 42 days. Drug concentrations in blood, brown and white adipose tissue, liver, and brain were measured up to 35 weeks postdose. During dosing, a 3- to 9-fold accumulation in 72-hour postdose blood concentrations of anacetrapib was observed. Drug concentrations in white adipose tissue accumulated ∼20- to 40-fold, whereas 10- to 17-fold accumulation occurred in brown adipose and approximately 4-fold in liver. Brain levels were very low (<0.1 μM), and a trend of accumulation was not seen. The presence of CETP as well as adiposity seems to play a role in determining the blood concentrations of anacetrapib. The highest blood concentrations were observed in NFR DIO mice, whereas the lowest concentrations were seen in WT lean mice. In adipose and liver tissue, higher concentrations were seen in DIO mice, irrespective of the presence of CETP. This finding suggests that white adipose tissue serves as a potential depot and that disposition into adipose tissue governs the long-term kinetics of anacetrapib in vivo.409Giudicelli, J. F.; Richer, C.; Berdeaux, A. Preliminary assessment of flutiorex, a new anorectic drug, in man. Br. J. Clin. Pharmacol. 1976, 3, 113– 121, DOI: 10.1111/j.1365-2125.1976.tb00578.x[Crossref], [PubMed], [CAS], Google Scholar409https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE28XkvVOntbg%253D&md5=a6a920a7186d45a423130e8d549c2f26Preliminary assessment of flutiorex, a new anorectic drug, in manGiudicelli, J. F.; Richer, Christine; Berdeaux, A.British Journal of Clinical Pharmacology (1976), 3 (1), 113-21CODEN: BCPHBM; ISSN:0306-5251.Flutiorex-HCl (I-HCl) [59960-78-0] exerted a significant anorectic effect and was approx. twice as potent as fenfluramine-HCl [404-82-0] in healthy volunteers. I induces a definite α-adrenergic sympathomimetic stimulation as was shown by a rise in systolic blood pressure and the development of marked mydriasis. I was a central nervous system stimulant producing an increase in crit. flicker frequency. It did not, however, influence psychomotor coordination as reflected in the pursuit rotor test. Serial detns. of blood and urine levels of I and its deethylated metabolite, norflutiorex [52771-23-0], showed that I was rapidly absorbed and deethylated, accumulated in large quantities in the tissues and, like its metabolite, was excreted in the urine in very small quantities. Blood levels of norflutiorex appear to remain elevated longer than those of I.410Silverstone, T.; Fincham, J.; Plumley, J. An evaluation of the anorectic activity in man of a sustained release formulation of tiflorex. Br. J. Clin. Pharmacol. 1979, 7, 353– 356, DOI: 10.1111/j.1365-2125.1979.tb00945.x[Crossref], [PubMed], [CAS], Google Scholar410https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1MXktlaqu7Y%253D&md5=51cfe71c0cc5b2e6fd345334337b775cAn evaluation of the anorectic activity in man of a sustained release formulation of tiflorexSilverstone, T.; Fincham, Jill; Plumley, JillBritish Journal of Clinical Pharmacology (1979), 7 (4), 353-6CODEN: BCPHBM; ISSN:0306-5251.In healthy female volunteers, a redn. in hunger following a slow release form of tiflorex (I) [53993-67-2] (20 mg, orally) was obsd. some 5-7 h after administration; no significant redn. in hunger occurred after placebo. A significant redn. in food intake occurred at a time corresponding to, and slightly later than, the subjective anorectic effect. I produced no effect on pulse rate or blood pressure, but it did induce some mydriasis. There was no evidence of CNS stimulation after I, and the drug had no influence on flicker fusion frequency or psychomotor performance. A redn. in subjective arousal was obsd. after 3 h, but I caused no other significant change in subjective measures of arousal or mood. Only slight sleep disturbance was reported; subjective reports of headache were more frequent after drug administration than after placebo, occurring in 7 out of 12 active drug exposures. Side effects were noted on 11 occasions after I, compared with 5 occasions after placebo.411Benoit, E.; Cresteil, T.; Riviere, J. L.; Delatour, P. Specific and enantioselective sulfoxidation of an aryl-trifluoromethyl sulfide by rat liver cytochromes P-450. Drug Metab. Dispos. 1992, 20, 877– 881[PubMed], [CAS], Google Scholar411https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXhtFKit7o%253D&md5=4f83b94031598759d705833a9b3514c4Specific and enantioselective sulfoxidation of an aryl-trifluoromethyl sulfide by rat liver cytochromes P-450Benoit, Etienne; Cresteil, Thierry; Riviere, Jean Louis; Delatour, PaulDrug Metabolism and Disposition (1992), 20 (6), 877-81CODEN: DMDSAI; ISSN:0090-9556.Evidence based on thermal stability and enzyme inhibition data suggests that the sulfoxidn. of the drug toltrazuril by rat liver microsomes is catalyzed by different cytochromes P 450. Pretreatment of rats by different inducers (phenobarbital, 3-methylcholanthrene, dexamethasone, and triacetyloleanodomycin) results in a 2.1-, 2.6-, 2.9-, and 1.8-fold increase, resp., in the rate of sulfoxidn. The highest increase (8.4-fold) was obsd. after treatment of microsomes from triacetyloleandomycin-treated animals by potassium ferricyanide. Castration and aging also modify the sulfoxidase activity. The relative rate of formation of the two toltrazuril enantiomers [(A)- and (B)-sulfoxides] depends on the source of the microsomes, suggesting that different cytochromes P 450 have different stereoselectivities.412Benoit, E.; Buronfosse, T.; Delatour, P. Effect of cytochrome P-450 1A induction on enantioselective metabolism and pharmacokinetics of an aryltrifluoromethyl sulfide in the rat. Chirality 1994, 6, 372– 377, DOI: 10.1002/chir.530060503[Crossref], [PubMed], [CAS], Google Scholar412https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXjs1GksA%253D%253D&md5=87b0af36f97233f6e2490d52f70bd250Effect of cytochrome P-450 1A induction on enantioselective metabolism and pharmacokinetics of an aryltrifluoromethyl sulfide in the ratBenoit, Etienne; Buronfosse, Thierry; Delatour, PaulChirality (1994), 6 (5), 372-7CODEN: CHRLEP; ISSN:0899-0042.The pharmacokinetics of the antiparasitic drug toltrazuril (1-methyl-3-[3-methyl-4-[4-[trifluoromethyl]thio]phenoxy]phenyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione) were studied in the rat following pretreatment with 3-methylcholanthrene, an inducer of rat liver cytochrome P 450 1A. The induction markedly modified the pharmacokinetics of the compd., leading to a decrease in the AUC value for toltrazuril sulfoxide. The results were explained on the basis of previous results from the authors lab. relating to the product enantioselectivity of the formation of the sulfoxide and the substrate enantioselectivity of the subsequent formation of the sulfone.413Mas-Chamberlin, C.; Gillet, G.; André, J.; Gomeni, R.; Dring, L. G.; Morselli, P. L. The metabolism and kinetics of tiflorex in the rat. Drug Metab. Dispos. 1981, 9, 150– 155[PubMed], [CAS], Google Scholar413https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXktVSitbo%253D&md5=24341578b2f862cfa323177c402f5cd2The metabolism and kinetics of tiflorex in the ratMas-Chamberlin, Claire; Gillet, Gerard; Andre, Joelle; Gomeni, Roberto; Dring, L. Graham; Morselli, Paolo L.Drug Metabolism and Disposition (1981), 9 (2), 150-5CODEN: DMDSAI; ISSN:0090-9556.14C-labeled tiflorex-HCl (I) [59960-78-0] given either orally or i.v. to male rats (10 mg/kg; 250 μCi) was well absorbed orally; >70% of the dose was excreted in the urine in the 1st 48 h after dosing by either route of administration. Part of the dose (10%) was excreted in the feces after i.v. administration; biliary excretion may be a route of elimination. The major route of metab. was S-oxidn. to give the sulfoxides and sulfones of tiflorex (7% each) and nortiflorex [52771-24-1] which were excreted together with the unchanged drug (1%) in the 0- to 48-h urine. Drug was rapidly absorbed orally, max. levels was attained within 30 min. The plasma half-life for the elimination phase of the unchanged drug was relatively long (7.5 h) compared with the metabolites (2.5 h) with the exception of nortiflorex sulfone [82560-96-1] (9.8 h) and 2 as yet unidentified metabolites which had half-lives in excess of 24 h. The latter 3 compds. were responsible for the relatively long plasma half-life of total radioactivity (∼13 h). The ratio of the areas under the plasma curve for unchanged drug indicated a low bioavailability (30%). Apparently, the predominant route of metab. of this group of compds. in the rat, p-hydroxylation, was blocked by the trifluoromethylthio group, with consequent emphasis on S-oxidn.414Karadzovska, D.; Seewald, W.; Browning, A.; Smal, M.; Bouvier, J.; Giraudel, J. M. Pharmacokinetics of monepantel and its sulfone metabolite, monepantel sulfone, after intravenous and oral administration in sheep. J. Vet. Pharmacol. Ther. 2009, 32, 359– 367, DOI: 10.1111/j.1365-2885.2008.01052.x[Crossref], [PubMed], [CAS], Google Scholar414https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtValtbjM&md5=96efb9456a5d61f4b1321ecd7ad08e0aPharmacokinetics of monepantel and its sulfone metabolite, monepantel sulfone, after intravenous and oral administration in sheepKaradzovska, D.; Seewald, W.; Browning, A.; Smal, M.; Bouvier, J.; Giraudel, J. M.Journal of Veterinary Pharmacology and Therapeutics (2009), 32 (4), 359-367CODEN: JVPTD9; ISSN:0140-7783. (Wiley-Blackwell)The pharmacokinetic properties of the developmental Amino-Acetonitrile Deriv. (AAD), monepantel and its sulfone metabolite, monepantel sulfone were investigated in sheep following i.v. and oral administrations. The sulfone metabolite was rapidly formed and predominated over monepantel 4 h after dosing, irresp. of the route of administration. The steady-state vol. of distribution, total body clearance and mean residence time of monepantel were 7.4 L/kg, 1.49 L/(kg/h) and 4.9 h, resp. and 31.2 L/kg, 0.28 L/(kg/h) and 111 h, resp. for monepantel sulfone. The overall bioavailability of monepantel was 31%, but it was demonstrated that approx. the same amt. of monepantel sulfone was produced whether monepantel was given i.v. or orally (AUC(0-∞) oral/AUC(0-∞) i.v. of 94% for monepantel sulfone), making oral administration a very efficient route of administration for monepantel in terms of the amt. of sulfone metabolite generated. Because monepantel sulfone is the main chem. entity present in sheep blood after monepantel administration and because it is also an active metabolite, its pharmacokinetic properties are of primary importance for the interpretation of future residue and efficacy studies. Overall, these pharmacokinetic data aid in the evaluation of monepantel as an oral anthelmintic in sheep.415Lim, J. H.; Kim, M. S.; Hwang, Y. H.; Song, I. B.; Park, B. K.; Yun, H. I. Pharmacokinetics of toltrazuril and its metabolites, toltrazuril sulfoxide and toltrazuril sulfone, after a single oral administration to pigs. J. Vet. Med. Sci. 2010, 72, 1085– 1087, DOI: 10.1292/jvms.09-0524[Crossref], [PubMed], [CAS], Google Scholar415https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtFKrtLrE&md5=cfca7a5e60162cfa3c525d5cb3b05980Pharmacokinetics of toltrazuril and its metabolites, toltrazuril sulfoxide and toltrazuril sulfone, after a single oral administration to pigsLim, Jong-Hwan; Kim, Myoung-Seok; Hwang, Youn-Hwan; Song, In-Bae; Park, Byung-Kwon; Yun, Hyo-InJournal of Veterinary Medical Science (2010), 72 (8), 1085-1087CODEN: JVMSEQ; ISSN:0916-7250. (Japanese Society of Veterinary Science)Toltrazuril (TZR) is a triazine-based antiprotozoal agent. Following a single oral administration of TZR at 10 and 20 mg/kg to male pigs, the mean TZR concn. in plasma peaked at 4.24 and 8.18 μg/mL at 15.0 and 12.0 h post-dose, resp. TZR absorbed was rapidly converted to the short-lived intermediary metabolite toltrazuril sulfoxide (TZR-SO), and then metabolized to the reactive toltrazuril sulfone (TZR-SO2). TZR-SO2 was actually more slowly eliminated, with av. half-lives of 231 and 245 h, compared with TZR (48.7 and 68.9 h) or TZR-SO (51.9 and 53.2 h) in the 10 and 20 mg/kg groups, resp. This study demonstrates that TZR metabolizes to TZR-SO2 having a long-terminal half-life, enabling the persistent clin. efficacy in the treatment of I. suis infection. In contrast, special consideration should be given to the residual of TZR-SO2.416Ghiazza, C.; Billard, T.; Dickson, C.; Tlili, A.; Gampe, C. M. Chalcogen OCF3 isosteres modulate drug properties without introducing inherent liabilities. ChemMedChem 2019, 14, 1586– 1589, DOI: 10.1002/cmdc.201900452[Crossref], [PubMed], [CAS], Google Scholar416https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1WisrjM&md5=d711d2096dfa2ed308a365e03e67e52cChalcogen OCF3 Isosteres Modulate Drug Properties without Introducing Inherent LiabilitiesGhiazza, Clement; Billard, Thierry; Dickson, Callum; Tlili, Anis; Gampe, Christian M.ChemMedChem (2019), 14 (17), 1586-1589CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)The synthesis of SCF3 as well as SeCF3 isosteres of two OCF3-contg. drugs was achieved through visible light and copper-catalyzed processes. Herein, we show that chalcogen replacement modulates physicochem. and ADME properties without introducing intrinsic liabilities. The SCF3 and SeCF3 groups are more lipophilic than their oxygen counterpart; however, microsomal stability is unchanged, indicating that these mol. changes may be beneficial for in vivo half-life. Enabled by modern synthetic methods, we present the chalcogen-CF3 groups as potential key players for future fluorinated pharmaceuticals.417Yu, L.; Ternansky, R. J.; Victoria, E. J.; Chang, J.; Coutts, S. M. The structure-activity relationships of a series of suicide inhibitors of phospholipase A2. Bioorg. Med. Chem. Lett. 1998, 8, 2129– 2132, DOI: 10.1016/S0960-894X(98)00378-3[Crossref], [PubMed], [CAS], Google Scholar417https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXmtVKrur4%253D&md5=b83cf378d87ef0123bec65340f02787fThe structure-activity relationships of a series of suicide inhibitors of phospholipase A2Yu, Lin; Ternansky, Robert J.; Victoria, Edward J.; Chang, Julia; Coutts, Stephen M.Bioorganic & Medicinal Chemistry Letters (1998), 8 (16), 2129-2132CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science Ltd.)A series of mechanism-based inhibitors of phospholipase A2 (SIBLINKS) were synthesized. These new SIBLINKS are phospholipid analogs that contain a para-substituted Ph 3,3-dimethylglutaryl group in the place of the sn-2 acyl chain. The effect of the Ph leaving group on inhibitory activity was studied by varying the electron-withdrawing ability of the para-substituted group. A strong correlation was obsd. between the leaving group potential of the suicide inhibitor and the inhibitory activity of the deriv. toward cobra venom phospholipase A2.418Pearce, R. E.; Leeder, J. S.; Kearns, G. L. Biotransformation of fluticasone: in vitro characterization. Drug Metab. Dispos. 2006, 34, 1035– 1040, DOI: 10.1124/dmd.105.009043[Crossref], [PubMed], [CAS], Google Scholar418https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XlsVOnuro%253D&md5=f6a3f266bf4d82a2039fb3bff1fdc69bBiotransformation of Fluticasone: in vitro characterizationPearce, Robin E.; Leeder, J. Steven; Kearns, Gregory L.Drug Metabolism and Disposition (2006), 34 (6), 1035-1040CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)Fluticasone propionate (FTP) is a synthetic trifluorinated glucocorticoid with potent anti-inflammatory action that is commonly used in patients with asthma. After oral or intranasal administration, FTP undergoes rapid hepatic biotransformation; the principal metabolite formed is a 17β-carboxylic acid deriv. (M1). M1 formation has been attributed largely to cytochrome P 450 3A4 (CYP3A4); however, there are no published data that confirm this assertion. Hence, in vitro studies were conducted to det. the role that human P450s play in the metab. of FTP. Consistent with in vivo data, human liver microsomes catalyzed the formation of a single metabolite (M1) at substrate concns. ≤10 μM (mean plasma Cmax = 1 nM). Under these conditions, the kinetics of M1 formation in human liver microsomes were consistent with those of a single enzyme (Km ≃ 5 μM). Formation of M1 correlated significantly (r > 0.95) with CYP3A4/5 activities in a panel of human liver microsomes (n = 14) and was markedly impaired by the CYP3A inhibitor ketoconazole (>94%) but not by inhibitors of other P 450 enzymes (≤10%). Studies with a panel of cDNA-expressed enzymes revealed that M1 formation was catalyzed primarily by CYP3A enzymes at FTP concns. ≤1 μM. M1 formation was catalyzed by P450s 3A4, 3A5, and 3A7; in vitro intrinsic clearance values (Vmax/Km) were comparable for all three CYP3A enzymes. These results suggest that at pharmacol. relevant concns., biotransformation of FTP to M1 is mediated predominantly by CYP3A enzymes in the liver.419Westphal, M. V.; Wolfstadter, B. T.; Plancher, J. M.; Gatfield, J.; Carreira, E. M. Evaluation of tert-butyl isosteres: case studies of physicochemical and pharmacokinetic properties, efficacies, and activities. ChemMedChem 2015, 10, 461– 469, DOI: 10.1002/cmdc.201402502[Crossref], [PubMed], [CAS], Google Scholar419https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtlWis7w%253D&md5=06f976396f0ec1bdb7a7f93fdd3808f3Evaluation of tert-Butyl isosteres: case studies of physicochemical and pharmacokinetic properties, efficacies, and activitiesWestphal, Matthias V.; Wolfstaedter, Bernd T.; Plancher, Jean-Marc; Gatfield, John; Carreira, Erick M.ChemMedChem (2015), 10 (3), 461-469CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)The tert-Bu group is a common motif in medicinal chem. Its incorporation into bioactive compds. is often accompanied by unwanted property modulation, such as increased lipophilicity and decreased metabolic stability. Several alternative substituents are available for the drug discovery process. Herein, physicochem. data of two series of drug analogs of bosentan and vercirnon are documented as part of a comparative study of tert-Bu, pentafluorosulfanyl, trifluoromethyl, bicyclo[1.1.1]pentyl, and cyclopropyl-trifluoromethyl substituents.420Sowaileh, M. F.; Hazlitt, R. A.; Colby, D. A. Application of the pentafluorosulfanyl group as a bioisosteric replacement. ChemMedChem 2017, 12, 1481– 1490, DOI: 10.1002/cmdc.201700356[Crossref], [PubMed], [CAS], Google Scholar420https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsV2ju7fK&md5=d7ebb1fd3f7f0f380f16608a16ffefc6Application of the Pentafluorosulfanyl Group as a Bioisosteric ReplacementSowaileh, Munia F.; Hazlitt, Robert A.; Colby, David A.ChemMedChem (2017), 12 (18), 1481-1490CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)The success of fluorinated mols. in drug design has led medicinal chemists to search for new fluorine-contg. substituents. A major recently developed group is the pentafluorosulfanyl group. This group is stable under physiol. conditions and displays unique phys. and chem. properties. There are currently few synthetic methods to install the SF5 group, yet efforts to integrate this group into lead optimization continue unabated. Typically, the SF5 group has been used as a replacement for trifluoromethyl, tert-Bu, halogen, or nitro groups. In this review, the use of the SF5 group as a bioisosteric replacement for each of these three functionalities is compared and contrasted across various groups of biol. active mols. The organization and presentation of these data should be instructive to medicinal chemists considering to design synthetic strategies to access SF5-substituted mols.421Hansch, C.; Muir, R. M.; Fujita, T.; Maloney, P. P.; Geiger, F.; Streich, M. The correlation of biological activity of plant growth regulators and chloromycetin derivatives with Hammett constants and partition coefficients. J. Am. Chem. Soc. 1963, 85, 2817– 2824, DOI: 10.1021/ja00901a033[ACS Full Text], [CAS], Google Scholar421https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF3sXksFKhtbk%253D&md5=ce902ea696b325cb22b74d58c9fcdd6cThe correlation of biological activity of plant growth regulators and chloromycetin derivatives with Hammett constants and partition coefficientsHansch, Corwin; Muir, Robert M.; Fujita, Toshio; Maloney, Peyton P.; Geiger, Fred; Streich, MargaretJournal of the American Chemical Society (1963), 85 (18), 2817-24CODEN: JACSAT; ISSN:0002-7863.An equation using 2 exptl. based variables, σ and π, was developed for correlating the effect of a given substituent on the biological activity of a parent compd.; is the Hammett substituent const. and π is an analogous const. representing the difference in the logarithms of the partition coeffs. of the substituted and unsubstituted compds. (π = log Px - log PH). The value of this equation was tested on 2 systems of biol. active mols.: the phenoxyacetic acids and chloromycetin analogs. By using π and a it becomes possible to disentangle three of the most important parameters governing the biol. activity of org. compns.: steric, electronic, and rate of penetration.422Welch, J. T.; Lim, D. S. The synthesis and biological activity of pentafluorosulfanyl analogs of fluoxetine, fenfluramine, and norfenfluramine. Bioorg. Med. Chem. 2007, 15, 6659– 6666, DOI: 10.1016/j.bmc.2007.08.012[Crossref], [PubMed], [CAS], Google Scholar422https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVCisbfP&md5=5aa869f37f1c306c1879cbb75aa251fbThe synthesis and biological activity of pentafluorosulfanyl analogs of fluoxetine, fenfluramine, and norfenfluramineWelch, John T.; Lim, Dong SungBioorganic & Medicinal Chemistry (2007), 15 (21), 6659-6666CODEN: BMECEP; ISSN:0968-0896. (Elsevier Ltd.)The trifluoromethyl group of fluoxetine, fenfluramine, and norfenfluramine was substituted by the pentafluorosulfanyl group. On examn. of the efficacy of the pentafluorosulfanyl contg. compds. as inhibitors of 5-hydroxytryptamine receptors, it was found that substitution could lead to enhanced selectivity and in the case of the pentafluorosulfanyl analog of fenfluramine (I) it significantly enhanced potency against the 5-HT2b, 5-HT2c, and 5-HT6 receptors.423Wipf, P.; Mo, T.; Geib, S. J.; Caridha, D.; Dow, G. S.; Gerena, L.; Roncal, N.; Milner, E. E. Synthesis and biological evaluation of the first pentafluorosulfanyl analogs of mefloquine. Org. Biomol. 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Initial bioactivity studies reveal effects as ion channel modulators and inhibitory activities on aldo-keto reductase 1C3 (AKR1C3) as well as COX-1 and COX-2.425Zhang, Y.; Wang, Y.; He, C.; Liu, X.; Lu, Y.; Chen, T.; Pan, Q.; Xiong, J.; She, M.; Tu, Z.; Qin, X.; Li, M.; Tortorella, M. D.; Talley, J. J. Pentafluorosulfanyl-substituted benzopyran analogues as new cyclooxygenase-2 inhibitors with excellent pharmacokinetics and efficacy in blocking inflammation. J. Med. 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The pentafluorosulfanyl compds. showed both potency and selectivity for COX-2 and demonstrated efficacy in several murine models of inflammation and pain. More interestingly, one of the compds., I, revealed exceptional efficacy in the adjuvant induced arthritis (AIA) model, achieving an ED50 as low as 0.094 mg/kg. In addn., the pharmacokinetics of compd. I in rat revealed a half-life in excess of 12 h and plasma drug concns. well above its IC90 for up to 40 h. When I was dosed just two times a week in the AIA model, efficacy was still maintained. Overall, drug I and other analogs are suitable candidates that merit further investigation for the treatment of inflammation and pain as well as other diseases where COX-2 and PGE2 play a role in their etiol.426Phillips, M. A.; Lotharius, J.; Marsh, K.; White, J.; Dayan, A.; White, K. L.; Njoroge, J. W.; El Mazouni, F.; Lao, Y.; Kokkonda, S.; Tomchick, D. R.; Deng, X.; Laird, T.; Bhatia, S. N.; March, S.; Ng, C. L.; Fidock, D. A.; Wittlin, S.; Lafuente-Monasterio, M.; Benito, F. J.; Alonso, L. M.; Martinez, M. S.; Jimenez-Diaz, M. B.; Bazaga, S. F.; Angulo-Barturen, I.; Haselden, J. N.; Louttit, J.; Cui, Y.; Sridhar, A.; Zeeman, A. M.; Kocken, C.; Sauerwein, R.; Dechering, K.; Avery, V. M.; Duffy, S.; Delves, M.; Sinden, R.; Ruecker, A.; Wickham, K. S.; Rochford, R.; Gahagen, J.; Iyer, L.; Riccio, E.; Mirsalis, J.; Bathhurst, I.; Rueckle, T.; Ding, X.; Campo, B.; Leroy, D.; Rogers, M. J.; Rathod, P. K.; Burrows, J. N.; Charman, S. A. A long-duration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria. Sci. Transl. Med. 2015, 7, 296ra111, DOI: 10.1126/scitranslmed.aaa6645427McCarthy, J. S.; Lotharius, J.; Ruckle, T.; Chalon, S.; Phillips, M. A.; Elliott, S.; Sekuloski, S.; Griffin, P.; Ng, C. L.; Fidock, D. A.; Marquart, L.; Williams, N. S.; Gobeau, N.; Bebrevska, L.; Rosario, M.; Marsh, K.; Mohrle, J. J. Safety, tolerability, pharmacokinetics, and activity of the novel long-acting antimalarial DSM265: a two-part first-in-human phase 1a/1b randomised study. Lancet Infect. Dis. 2017, 17, 626– 635, DOI: 10.1016/S1473-3099(17)30171-8[Crossref], [PubMed], [CAS], Google Scholar427https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXltlWmur8%253D&md5=144cece3cf70a64af921f7f37df7c72dSafety, tolerability, pharmacokinetics, and activity of the novel long-acting antimalarial DSM265: a two-part first-in-human phase 1a/1b randomised studyMcCarthy, James S.; Lotharius, Julie; Ruckle, Thomas; Chalon, Stephan; Phillips, Margaret A.; Elliott, Suzanne; Sekuloski, Silvana; Griffin, Paul; Ng, Caroline L.; Fidock, David A.; Marquart, Louise; Williams, Noelle S.; Gobeau, Nathalie; Bebrevska, Lidiya; Rosario, Maria; Marsh, Kennan; Mohrle, Jorg J.Lancet Infectious Diseases (2017), 17 (6), 626-635CODEN: LIDABP; ISSN:1473-3099. (Elsevier Ltd.)DSM265 is a novel antimalarial that inhibits plasmodial dihydroorotate dehydrogenase, an enzyme essential for pyrimidine biosynthesis. We investigated the safety, tolerability, and pharmacokinetics of DSM265, and tested its antimalarial activity. Healthy participants aged 18-55 years were enrolled in a two-part study: part 1, a single ascending dose (25-1200 mg), double-blind, randomised, placebo-controlled study, and part 2, an open-label, randomised, active-comparator controlled study, in which participants were inoculated with Plasmodium falciparum induced blood-stage malaria (IBSM) and treated with DSM265 (150 mg) or mefloquine (10 mg/kg). Primary endpoints were DSM265 safety, tolerability, and pharmacokinetics. Randomisation lists were created using a validated, automated system. Both parts were registered with the Australian New Zealand Clin. Trials Registry, no. ACTRN12613000522718 (part 1) and no. ACTRN12613000527763 (part 2). In part 1, 73 participants were enrolled between Apr. 12, 2013, and July 14, 2015 (DSM265, n = 55; placebo, n = 18). In part 2, nine participants were enrolled between Sept 30 and Nov 25, 2013 (150 mg DSM265, n = 7; 10 mg/kg mefloquine, n = 2). In part 1, 117 adverse events were reported; no drug-related serious or severe events were reported. The most common drug-related adverse event was headache. The mean DSM265 peak plasma concn. (Cmax) ranged between 1310 ng/mL and 34 800 ng/mL and was reached in a median time (tmax) between 1.5 h and 4 h, with a mean elimination half-life between 86 h and 118 h. In part 2, the log10 parasite redn. ratio at 48 h in the DSM265 (150 mg) group was 1.55 (95% CI 1.42-1.67) and in the mefloquine (10 mg/kg) group was 2.34 (2.17-2.52), corresponding to a parasite clearance half-life of 9.4 h (8.7-10.2) and 6.2 h (5.7-6.7), resp. The median min. inhibitory concn. of DSM265 in blood was estd. as 1040 ng/mL (range 552-1500), resulting in a predicted single efficacious dose of 340 mg. Parasite clearance was significantly faster in participants who received mefloquine than in participants who received DSM265 (p<0.0001). The good safety profile, long elimination half-life, and antimalarial effect of DSM265 supports its development as a partner drug in a single-dose antimalarial combination treatment. Wellcome Trust, UK Department for International Development, Global Health Innovative Technol. Fund, Bill & Melinda Gates Foundation.
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Abstract
Figure 1
Figure 1. Fluorine-containing drugs approved by the U.S. Food and Drug Administration through the end of 2019.
Scheme 1
Scheme 1. (S)-γ-Fluoroleucine (1) Converts Spontaneously to Amino Lactone 2 with Loss of HFFigure 2
Figure 2. Fluoroacetic acid (5) and known metabolic precursors 6–12.
Scheme 2
Scheme 2. Biosynthesis of 5 and 18 and (Inset) Details of the Transition State as Proposed by Theoretical, Structural, and Kinetic StudiesScheme 3
Scheme 3. Biochemical Reactions of Native and Fluorinated Substrates Catalyzed by Aconitaseaa(A) Dehydration of citric acid (19) to form 20 is followed by rehydration to give isocitric acid (21). (B) Compound 5 is converted to fluoroacetyl-CoA (22), which reacts by a Claisen condensation with 23 to give (−)-erythro-(2R,3R)-2-fluorocitric acid (24) in a stereospecific manner. Dehydration of 24 to give 25 is followed by a flip in the active site as a prelude to the addition of H2O. This addition produces 4-hydroxy-trans-aconitate (26), a potent and tight-binding inhibitor of aconitase that represents the ultimate toxicant produced by the biochemical transformation of 5.
Scheme 4
Scheme 4. Metabolism of (+)-erythro-(2S,3S)-2-Fluorocitric Acid (27) by Aconitaseaa(+)-erythro-(2S,3S)-2-Fluorocitric acid (27) is subject to elimination of H2O to afford 28, which flips in the enzyme active site to set the stage for the addition of H2O to produce 29. This compound collapses with loss of HF to give oxalosuccinic acid (30), which decarboxylates to afford α-ketoglutaric acid (31).
Scheme 5
Scheme 5. Potential Mechanism for the Metabolic Activation of 1,3-Difluoro-2-propanol (32) to Form Fluoroacetic Acid CoA EsteraaThis process is hypothesized to proceed via oxidation of 32 by ALDH to give 1,3-difluoroacetone (33) followed by a Baeyer–Villiger-type process to generate ester 12′. This ester would be anticipated to convert readily to 5 by hydrolysis or otherwise undergo esterase-mediated cleavage to release 5, formaldehyde, and fluoride.
Figure 3
Figure 3. Example of the tactical deployment of fluorine to modulate the basicity in piperidine-based KSP inhibitors.
Scheme 6
Scheme 6. Major Metabolic Pathway for 35 in RLM and HepatocytesScheme 7
Scheme 7. Enzymatic Baeyer–Villiger Oxidation of Z-Phe-Ala-CH2F (40)Scheme 8
Scheme 8. Covalent Inhibition of ATG4B by the Fluorinated Peptide Mimetic 45 to Afford Alkylated Enzyme 46Scheme 9
Scheme 9. Biotransformation of 49aa(a) 5-Fluorouracil (49) is a substrate of both orotate phosphoribosyl transferase, which catalyzes the addition of a phosphorylated ribose moiety to yield 55, and dihydropyrimidine dehydrogenase, which carries out the initial step in the catabolic pathway that mediates the disposition of the drug. (b) The triphosphate metabolite 56 may be incorporated into DNA, leading to downstream single-strand breaks. (c) The monophosphate 57 reacts with thymidylate synthase and 5,10-methylene tetrahydrofolate, forming the covalent inactivated enzyme complex 58. (d) The multistep catabolic sequence proceeds through a postulated carbon–carbon bond cleavage and decarboxylation, culminating in the formation of 5, in which the acid group may be derived from either C-4 or C-6 of the starting material.
Scheme 10
Scheme 10. Metabolism of Capecitabine (59) to 49Figure 4
Figure 4. Optimization of HCV NS4B inhibitors to avoid the production of 33, a metabolic precursor to 5.
Scheme 11
Scheme 11. Compounds Containing N-Trifluoroethyl Groups May Be Metabolized In Vivo to Release 71, Which Causes Testicular ToxicityScheme 12
Scheme 12. Mechanism of Defluorination of 5 by Mammalian and Bacterial Defluorinase EnzymesScheme 13
Scheme 13. Metabolic Pathways of 5, 10, and 11 in RodentsScheme 14
Scheme 14. Metabolic Pathways of HCFCs 83–85aaThe metabolism of 83–85 proceeds in each case through an aldehyde intermediate, which is subject to differential metabolism depending on the pattern of halogen substitution (X′, X″, and X‴).
Scheme 15
Scheme 15. Metabolism of HFC-245fa (86)Scheme 16
Scheme 16. Metabolic Activation of 94, Which is Metabolized by P450 along (a) Reductive and (b) Oxidative Pathwaysaa(a) The reductive pathway involves a one-electron reduction with loss of bromine to afford a radical, which either abstracts a hydrogen atom prior to exhalation or is further reduced. The product of reduction is an unstable 1-chloro-2,2,2-trifluoroethyl carbanion intermediate that eliminates fluoride to give 96, which is also exhaled. (b) The oxidative pathway involves oxidation and loss of HBr to give 97, which is readily hydrolyzed to give 67.
Scheme 17
Scheme 17. Metabolism of 98Scheme 18
Scheme 18. Metabolism of 104, a Base-Catalyzed Degradation Product of 98, in Rats and HumansScheme 19
Scheme 19. In Vitro Metabolism of a Homologous Series of [18F]fluoroalkylbiphenyl DerivativesaaBenzylic oxidation of 114 releases fluoride immediately, whereas 117 and 121 require successive benzylic oxidations via the intermediate alcohols 118 and 122, respectively, to afford the ketones 119 and 123. Since the release of fluoride is faster from the intermediate derived from 121 than that from 117, the latter is deemed to have the best overall properties. This compound is oxidized in successive steps to 118 and the α-fluoro ketone 119, which is believed to undergo spontaneous but slow α-elimination (16% over 1 h) to give the corresponding enol. This enol is tautomeric with the ketone 120.
Figure 5
Figure 5. 11β-Fluoroalkyl and 11β-fluoroalkoxy estrogen derivatives explored for their potential as PET imaging agents.
Figure 6
Figure 6. Structures of 11C-WAY-100635 (11C-129) and [18F]-derived analogues 130–135 explored as potential PET tracers. The asterisk denotes the position of the 11C label in 11C-129.
Scheme 20
Scheme 20. Metabolism of ((1R,2S,3R)-2,3-Difluorocyclohexyl)benzene (136) by C. elegansaaComplete conversion was obtained over 72 h at 28 °C. Compounds 137–139 represent the metabolites observed in the study, which included other polyfluorinated substrates in addition to 136. The main site of oxidation was at the benzyic carbon rather than at sites substituted by fluorine. The unexpected formation of 140 was not explained but serves as a reminder that fluorinated alkyl groups may not always be metabolically inert.
Figure 7
Figure 7. Influence of the pattern of difluorination on the physicochemical properties of the matched pair of indoles 141 and 142 and the HIF2α inhibitors 143 (PT2385) and 144 (PT2977). The solubilities of 141 and 142 were measured in aqueous buffer (pH 6.5), while clearance was measured in human liver microsomes and protein binding of 143 and 144 was measured in human plasma.
Scheme 21
Scheme 21. Fluticasone Furoate (145) and Flunisolide (146) Are Both Metabolized by Allylic Oxidation at C-6, Which Results in the Loss of Fluorineaa(A) Fluticasone furoate (145) undergoes abstraction of the axial 6-hydrogen by P450 with rebound oxidation, leading to 147. This intermediate eliminates fluoride to form the keto compound 148, which is reduced to the 6-hydroxy metabolite 149. While the configuration of 149 has not been determined, analogy to flunisolide (146), which is metabolized in a stereospecific manner (B), would suggest that both steroids likely undergo β-oxidation at this site.
Figure 8
Figure 8. Fluorinated alkenes can serve as isosteres of a ketone, ester, or amide (A) or a carbonyl moiety (B), depending on the specific structural arrangement.
Scheme 22
Scheme 22. Proposed Nucleophilic Substitution of 1,1,3,3,3-Pentafluoro-2-(trifluoromethyl)prop-1-ene (158) with NucleophilesFigure 9
Figure 9. Correlation among the reactivity of perfluorinated olefins, the stability of carbanion intermediates, and lethality.
Scheme 23
Scheme 23. Metabolic Activation of 160 by CYP2E1 to Generate Fluoroethylene Oxide (166), a Reactive Intermediate That Can Modify DNA Bases or Form Downstream ProductsScheme 24
Scheme 24. Proposed Metabolic Pathway of 157 Leading to Tissue-Selective ToxicityaaThe reaction of 157 with hepatic GSH generates S-(1,1,2,2-tetrafluoroethyl)GS (174), which is processed to S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (175) and excreted into bile. Following absorption, 175 undergoes β-lysis to form the thiolate intermediate 176 along with pyruvic acid and ammonia. Elimination of fluoride from 176 generates the reactive thionoacyl fluoride 177, which is hypothesized to be the ultimate toxicant.
Scheme 25
Scheme 25. Metabolism of 2-Bromo-2-chloro-1,1-difluoroethylene (182)Scheme 26
Scheme 26. Conversion of Squalene (192) to (3S)-2,3-Oxidosqualene (193) by Squalene Monooxygenase, the Initial Step in the Biosynthesis of SterolsScheme 27
Scheme 27. Proposed Mechanisms of Inhibition of SAHase by Neoplanocin A (201) and Fluoroneplanocin A (202)Scheme 28
Scheme 28. Inhibition of MAO B by Mofegiline (209)aaThe inhibition results from oxidation of the primary amine to the conjugated iminium 210, a Michael acceptor that reacts with N5 of the flavin coenzyme, forming the covalent adduct 211. Loss of fluoride from 211 results in the formation of a covalent complex (212).
Scheme 29
Scheme 29. Inactivation of SSAO/VAP-1 by Mofegiline (209)aaMofegiline is believed to condense with (2,4,5-trihydroxyphenyl)alanine quinone (214), the SSAO/VAP-1 cofactor, to form the quinone imine 215. Tautomerization of 215 affords the conjugated imine 216, which is intercepted by a proximal nucleophilic amino acid residue to give 217. This intermediate can eliminate fluoride, leading to the irreversibly inactivated enzyme 218.
Scheme 30
Scheme 30. Metabolic Pathways of Mofegiline (209) in Dogs and HumansScheme 31
Scheme 31. Proposed Mechanism for the Formation of 222 from 209 via Carbamic Acid 221Scheme 32
Scheme 32. Proposed Mechanism for the Formation of 220 from 209Scheme 33
Scheme 33. Proposed Mechanism of Irreversible (Path a) and Reversible (Path b) Inhibition of GABAAT by the GABA Mimic Vigabatrin (229)Scheme 34
Scheme 34. Proposed Mechanism of Irreversible Inhibition of GABAAT by 228aaThe inhibition is initiated by formation of the Schiff base 234 with PLP (a). Arg192 engages the carboxylate moiety to anchor the inhibitor in the active site of the enzyme. Tautomerization (b) of 234 generates the Michael acceptor 236, which is subject to successive hydrolytic reactions (c and d) that liberate 2 equiv of fluoride to ultimately produce 238. Because of conformational restrictions, 236 reacts with H2O rather than the catalytic Lys329 to form an unstable difluorohydrin, which degrades to the acyl fluoride 237 and then to PLP-bound diacid 238. The nascent carboxylate in 238 is positioned to engage in a second electrostatic interaction with Arg445. Hydrolysis of 238 (e) releases PLP-derived amine 239 and (1S)-4-oxocyclopentane-1,3-dicarboxylate (240), which can spontaneously undergo decarboxylation (f) to give (S)-3-oxocyclopentane-1-carboxylate (241).
Scheme 35
Scheme 35. Proposed Mechanism of the Metabolism of OSI-930 (242)aaOSI-930 (242) undergoes single-electron oxidation by P450, creating a radical intermediate that exists in both nitrogen- and carbon-centered resonance forms (243) prior to recombination/rebound hydroxylation. The resulting hemiketal 244 loses CF3OH, which rapidly degrades to fluorophosgene (246) at room temperature, producing the transient quinone imine intermediate 245. Quinone 245 is then reduced to the observed hydroxyphenyl metabolite 247.
Scheme 36
Scheme 36. Proposed Metabolic Pathways to Explain the Formation of Metabolites Observed with the CRF Receptor Antagonist BMS-665053 (255)Scheme 37
Scheme 37. Displacement of the OCHF2 Group in 263 by a Biological ThiolScheme 38
Scheme 38. Metabolism of 271 by C. elegans, Which Catalyzes O-Demethylation of the Anisole Methyl Moiety to Afford the Hydrolytically Sensitive Product 272Scheme 39
Scheme 39. Two Potential Mechanisms by Which Lumacaftor (286) May Be Metabolized to the Catechol 290aa(A) Arene oxidation of 286 by P450 affords 287, which degrades via 288. The degradation pathway for 288 could occur by the direct departure of the phenolate with concomitant release of fluorophosgene (246) or via elimination of HF and hydrolysis of the carbonofluoridate product followed by decarboxylation. Collapse to o-quinone 289 is followed by reduction to the catechol 290, which is either excreted or sulfated. (B) Arene oxidation of 286 at an alternate site would afford epoxide intermediate 291, which could degrade hydrolytically to give 292. Rearrangement of this intermediate with loss of H2O and reduction by an overall mechanism analogous to that described above would afford catechol 290 via the o-quinone 289.
Scheme 40
Scheme 40. Mechanism-Based Glycosidase Inactivation by the Glycoside 293 Exhibiting a Difluorinated EtheraaThe substituted ether of the glycoside is displaced (294), generating 295 and the difluorinated alcohol 296, which decays quickly to the acyl fluoride 297. The acyl fluoride then reacts covalently with an amino acid residue in the active site, inactivating the enzyme (298), or diffuses away from the enzyme and is either hydrolyzed or captured by an adventitious nucleophile such as a protein.
Scheme 41
Scheme 41. Metabolic Pathway Elucidated for the DPP4 Inhibitors 302 and 303 in RLMScheme 42
Scheme 42. Alternative Epoxide-Based Metabolic Pathway Contemplated for the DPP4 Inhibitors 302 and 303 in RLMFigure 10
Figure 10. Relationships between structure and CYP3A inhibition for a series of azepane derivatives.
Scheme 43
Scheme 43. Potential Metabolic Pathways Giving Rise to CYP3A TDI from Difluorinated Azepane DerivativesaaProposed metabolic pathways that may result in TDI of P450 by 318: (a) Oxidation of the primary amine in two steps yields the nitroso derivative 326, which coordinates to the heme protein. (b) Oxidative deamination yields the ketone 327, which undergoes elimination to give the α,β-unsaturated carbonyl 328 and then nucleophilic attack by a biological thiol to give 329 and 330. (c) In the des-NH2 series, α-hydroxylation of the azepane ring to give 331 leads to conjugated imine 332 and then 333, which also undergoes nucleophilic attack to give 334.
Scheme 44
Scheme 44. Mechanism of Inhibition of Bacterial Alanine Racemase by d-Fluoroalanine (339)Scheme 45
Scheme 45. Mechanism of Inhibition of Bacterial Alanine Racemase by Difluoroalanine (341)Scheme 46
Scheme 46. Mechanism of Inhibition of Bacterial Alanine Racemase by Trifluoroalanine (342)Scheme 47
Scheme 47. Mechanism of Inhibition of Bacterial Alanine Racemase by Fluorovinylglycine (365)Scheme 48
Scheme 48. Mode of Inhibition of Amino Acid Decarboxylases by β-Fluorinated Amino Acid Derivatives 386Scheme 49
Scheme 49. Mode of Inhibition of Tryptophan Synthase by 394Scheme 50
Scheme 50. (a) E1cb Mechanism of HF Elimination from (R)-404; (b) E2 Mechanism of HF Elimination from (S)-404Figure 11
Figure 11. Conformations of (a) (R)-404 and (c) (S)-404 recognized initially by GABAAT and (b, c) the two low-energy conformations of (S)-404.
Scheme 51
Scheme 51. Proposed Mechanism of Defluorination of 410 by GABAAT and Further Metabolism to Give 416 and 417Scheme 52
Scheme 52. Metabolic Fate of 340Scheme 53
Scheme 53. Pyruvate Dehydrogenase Uses the Cofactor Thiamine Diphosphate (419) to Effect the Decarboxylative Degradation of 355Scheme 54
Scheme 54. Proposed Bioactivation of 430 to Give Electrophile 432 and Trapping by a Lys of Serum AlbuminScheme 55
Scheme 55. Aromatic Oxidation Accompanied by the NIH Shift of Fluorine in FluorobenzenesScheme 56
Scheme 56. Metabolism of 448, a Quinoxaline-Based Non-Nucleoside Inhibitor of HIV-1 Reverse TranscriptaseScheme 57
Scheme 57. Potential Metabolic Activation Pathways of the Hedgehog Pathway Inhibitor 452Scheme 58
Scheme 58. Proposed Mode of Inhibition of HCV NS5B Polymerase by 456 in HCV RepliconsScheme 59
Scheme 59. Proposed Bioactivation Pathway for the BACE-1 Inhibitor 464, a 5-Fluorinated Pyrimidine Derivative Associated with CYP3A4 TDIScheme 60
Scheme 60. Proposed Metabolic Pathways of the Pentafluorophenyl-Based DPP4 Inhibitor 470 to give 471 and 472 (the Regiochemistry of GSH in 471 and 472 Was Not Determined)Scheme 61
Scheme 61. Formation of N-Oxide Metabolites 501 and 502 from 500 In VivoFigure 12
Figure 12. Metabolic soft spots associated with 488 in liver microsomes.
Scheme 62
Scheme 62. Mechanism Proposed to Explain the Reaction of 513 with DDAHScheme 63
Scheme 63. Metabolism of 4-Chloropyridine-Based Spectinamide Derivatives 516 Catalyzed by GSTScheme 64
Scheme 64. Postulated Mechanism of Inactivation of Tyrosine Hydroxylase by 550 and 551Scheme 65
Scheme 65. Base-Catalyzed Reaction (Path a) and Trytophanase-Mediated Metabolism (Path b) of 552Scheme 66
Scheme 66. Mechanism-Based Inactivation of Neuraminidases by Sialoside Substrates Bearing Difluoromethylphenol GroupsFigure 13
Figure 13. Design of the fluorescent probe 561 for assessment of galactosidase activity.
Scheme 67
Scheme 67. Mechanism-Based Labeling of a Galactosidase by the Fluorescent Reagent 561Scheme 68
Scheme 68. Mechanism of Base-Catalyzed Hydrolysis of Difluoromethylimidazoles 566 and 567 to Afford the Corresponding Aldehydes 570 and 571Scheme 69
Scheme 69. Base-Catalyzed Hydrolysis of Phenol 572 to Give the Acid 575Scheme 70
Scheme 70. Mechanism of Photodegradation of the CF3 Moiety of 578Scheme 71
Scheme 71. Photodegradation of 581 under Simulated Sunlight in Deionized H2O to Give the Carboxylic Acid 582Scheme 72
Scheme 72. Photodegradation of 584, Which Is Produced In Vivo from the Prodrug 583Scheme 73
Scheme 73. Light-Catalyzed Polymerization of 589Scheme 74
Scheme 74. Use of Aryl CF3 Moieties in the Design of Latent Electrophiles, Which Can Be Generated by Irradiation with Light and Used to Covalently Modify Target ProteinsScheme 75
Scheme 75. Two Potential Mechanisms for Elimination of –CF3 from 604Scheme 76
Scheme 76. Substituent at the 2-Position of the Quinone (Methyl or Difluoromethyl) Affects the Chemical and Biochemical Pharmacological Profile of Antischistosomal CompoundsScheme 77
Scheme 77. Mechanism of Inhibition of Thymidylate Synthase by 614, the Monophosphate Metabolite of 613Scheme 78
Scheme 78. Oxidative Metabolism of the CF3S Substituent of 624 in RLM and Rats to Afford the Sulfoxides 626 and 627 and Sulfone 628aaThe absolute configurations of the sulfoxides 626 and 627 were not determined, and the assignments shown are arbitrary.
Scheme 79
Scheme 79. Substituent Effects on Phospholipase A2 Suicide Inhibition by 633Scheme 80
Scheme 80. Metabolism of the [(Fluoromethyl)thio]carbonyl Moiety of 145 by P450 to Afford the Carboxylic Acid 638References
ARTICLE SECTIONSThis article references 427 other publications.
- 1Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.; Meanwell, N. A. Applications of fluorine in medicinal chemistry. J. Med. Chem. 2015, 58, 8315– 8359, DOI: 10.1021/acs.jmedchem.5b00258[ACS Full Text], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1ajs7%252FK&md5=9995829a94a8c0b8d9fb0d21bdfd5a1dApplications of Fluorine in Medicinal ChemistryGillis, Eric P.; Eastman, Kyle J.; Hill, Matthew D.; Donnelly, David J.; Meanwell, Nicholas A.Journal of Medicinal Chemistry (2015), 58 (21), 8315-8359CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review with meta-anal. The role of fluorine in drug design and development is expanding rapidly as we learn more about the unique properties assocd. with this unusual element and how to deploy it with greater sophistication. The judicious introduction of fluorine into a mol. can productively influence conformation, pKa, intrinsic potency, membrane permeability, metabolic pathways, and pharmacokinetic properties. In addn., 18F has been established as a useful positron emitting isotope for use with in vivo imaging technol. that potentially has extensive application in drug discovery and development, often limited only by convenient synthetic accessibility to labeled compds. The wide ranging applications of fluorine in drug design are providing a strong stimulus for the development of new synthetic methodologies that allow more facile access to a wide range of fluorinated compds. In this review, we provide an update on the effects of the strategic incorporation of fluorine in drug mols. and applications in positron emission tomog.
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- 6Zhou, Y.; Wang, J.; Gu, Z.; Wang, S.; Zhu, W.; Acena, J. L.; Soloshonok, V. A.; Izawa, K.; Liu, H. Next generation of fluorine-containing pharmaceuticals, compounds currently in phase II-III clinical trials of major pharmaceutical companies: New structural trends and therapeutic areas. Chem. Rev. 2016, 116, 422– 518, DOI: 10.1021/acs.chemrev.5b00392[ACS Full Text], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVGms7o%253D&md5=e3d07d070fcf5b69a54f70bdba99acd4Next Generation of Fluorine-Containing Pharmaceuticals, Compounds Currently in Phase II-III Clinical Trials of Major Pharmaceutical Companies: New Structural Trends and Therapeutic AreasZhou, Yu; Wang, Jiang; Gu, Zhanni; Wang, Shuni; Zhu, Wei; Acena, Jose Luis; Soloshonok, Vadim A.; Izawa, Kunisuke; Liu, HongChemical Reviews (Washington, DC, United States) (2016), 116 (2), 422-518CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. We focus herein on a new collection of 51 fluorine-contg. mols. that either were approved after 2011 by the corresponding agencies or still are on clin. development. These compds. cover a large no. of therapeutic areas and, on this occasion, we decided to classify them according to the no. of fluorine atoms or fluorinated moieties. In each case, the structures and modes of action of these new drugs are revised, with a particular focus on the role of fluorine in the biol. and pharmaceutical properties, when appropriate. Finally, representative synthetic routes are highlighted.
- 7Wang, J.; Sanchez-Rosello, M.; Acena, J. L.; del Pozo, C.; Sorochinsky, A. E.; Fustero, S.; Soloshonok, V. A.; Liu, H. Fluorine in pharmaceutical industry: fluorine-containing drugs introduced to the market in the last decade (2001–2011). Chem. Rev. 2014, 114, 2432– 2506, DOI: 10.1021/cr4002879[ACS Full Text], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFegsb7I&md5=07faac419cd090503e82c6f00d05c338Fluorine in Pharmaceutical Industry: Fluorine-Containing Drugs Introduced to the Market in the Last Decade (2001-2011)Wang, Jiang; Sanchez-Rosello, Maria; Acena, Jose Luis; del Pozo, Carlos; Sorochinsky, Alexander E.; Fustero, Santos; Soloshonok, Vadim A.; Liu, HongChemical Reviews (Washington, DC, United States) (2014), 114 (4), 2432-2506CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A historic review. As expected from the fluorine position on the periodic table of elements, it possesses some extreme properties, in particular, ultimate electronegativity and oxidn. potential. Therefore,elemental fluorine can not be prepd. by chem. reaction, and its isolation in 1886 by Henri Moissan required scientific ingenuity and great personal courage. His historic effort earned him a Noble Prize (1906), and the developed electrolysis method is still in use for industrial prodn. of fluorine gas. However, further development of fluorine chem. was extremely sluggish, pursued by a handful of experts capable of handling the violent gas using specially designed lab. equipment. Industrial-scale prodn. of fluorochems. dates back to late 1930s. Currently, there are about 200 pharmaceuticals containingfluorine, including the 40 new compds. discussed in this review. One may agree that the contribution of the past decade indicates a significant 20% increase in the no. of fluorinated drugs on the market.
- 8Ilardi, E. A.; Vitaku, E.; Njardarson, J. T. Data-mining for sulfur and fluorine: an evaluation of pharmaceuticals to reveal opportunities for drug design and discovery. J. Med. Chem. 2014, 57, 2832– 2842, DOI: 10.1021/jm401375q[ACS Full Text], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFyksb%252FE&md5=b684f76341311979d0100ed68cffe714Data-Mining for Sulfur and Fluorine: An Evaluation of Pharmaceuticals To Reveal Opportunities for Drug Design and DiscoveryIlardi, Elizabeth A.; Vitaku, Edon; Njardarson, Jon T.Journal of Medicinal Chemistry (2014), 57 (7), 2832-2842CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A review. Among carbon, hydrogen, oxygen, and nitrogen, sulfur and fluorine are both leading constituents of the pharmaceuticals that comprise our medicinal history. In efforts to stimulate the minds of both the general public and expert scientist, statistics were collected from the trends assocd. with therapeutics spanning 12 disease categories (a total of 1969 drugs) from our new graphical montage compilation: disease focused pharmaceuticals posters. Each poster is a vibrant display of a collection of pharmaceuticals (including structural image, Food and Drug Administration (FDA) approval date, international nonproprietary name (INN), initial market name, and a color-coded subclass of function) organized chronol. and classified according to an assocn. with a particular clin. indication. Specifically, the evolution and structural diversity of sulfur and the popular integration of fluorine into drugs introduced over the past 50 years are evaluated. The presented qual. conclusions in this article aim to promote innovative insights into drug development.
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- 11Fujiwara, T.; O’Hagan, D. Successful fluorine-containing herbicide agrochemicals. J. Fluorine Chem. 2014, 167, 16– 29, DOI: 10.1016/j.jfluchem.2014.06.014[Crossref], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtFCgu7%252FM&md5=7fe60bc6faa07bfd629833ce8911c488Successful fluorine-containing herbicide agrochemicalsFujiwara, Tomoya; O'Hagan, DavidJournal of Fluorine Chemistry (2014), 167 (), 16-29CODEN: JFLCAR; ISSN:0022-1139. (Elsevier B.V.)A review. Of the herbicides licensed worldwide, currently around 25% contain at least one fluorine atom and many contain multiple fluorines in the form of difluoro- and trifluoromethyl groups. Fluorine-contg. compds. have made a significant contribution to the development of products for the agrochems. industry and many organofluorine entities have found stable market positions. In this review we highlight the most important fluorinated herbicides in terms of their global use. The compds. are grouped by mode of action. A synthesis route is described for each compd. although the synthesis presented may not actually be the industrial process.
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- 23Murphy, C. D.; Sandford, G. Recent advances in fluorination techniques and their anticipated impact on drug metabolism and toxicity. Expert Opin. Drug Metab. Toxicol. 2015, 11, 589– 599, DOI: 10.1517/17425255.2015.1020295[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXksFymt78%253D&md5=b5c542a9e9d7e3c246d4f24da4918f2bRecent advances in fluorination techniques and their anticipated impact on drug metabolism and toxicityMurphy, Cormac D.; Sandford, GrahamExpert Opinion on Drug Metabolism & Toxicology (2015), 11 (4), 589-599CODEN: EODMAP; ISSN:1742-5255. (Informa Healthcare)A review. Introduction: Fluorine's unique physicochem. properties make it a key element for incorporation into pharmacol. active compds. Its presence in a drug can alter a no. of characteristics that affect ADME-Tox, which has prompted efforts at improving synthetic fluorination procedures. Areas covered: This review describes the influence of fluorine on attributes such as potency, lipophilicity, metabolic stability and bioavailability and how the effects obsd. are related to the physicochem. characteristics of the element. Examples of more recently used larger scale synthetic methods for introduction of fluorine into drug leads are detailed and the potential for using biol. systems for fluorinated drug prodn. is discussed. Expert opinion: The synthetic procedures for carbon-fluorine bond formation largely still rely on decades-old technol. for the manufg. scale and new reagents and methods are required to meet the demands for the prepn. of structurally more complex drugs. The improvement of in vitro and computational methods should make fluorinated drug design more efficient and place less emphasis on approaches such as fluorine scanning and animal studies. The introduction of new fluorinated drugs, and in particular those that have novel fluorinated functional groups, should be accompanied by rigorous environmental assessment to det. the nature of transformation products that may cause ecol. damage.
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- 28Nodwell, M. B.; Yang, H.; Colovic, M.; Yuan, Z.; Merkens, H.; Martin, R. E.; Benard, F.; Schaffer, P.; Britton, R. 18)F-Fluorination of unactivated C-H bonds in branched aliphatic amino acids: Direct synthesis of oncological positron emission tomography imaging agents. J. Am. Chem. Soc. 2017, 139, 3595– 3598, DOI: 10.1021/jacs.6b11533[ACS Full Text], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjsVyqtrc%253D&md5=0a17fa3fdaa61c7b3c5e7dda1ea8975818F-Fluorination of unactivated C-H bonds in branched aliphatic amino acids: Direct synthesis of oncological positron emission tomography imaging agentsNodwell, Matthew B.; Yang, Hua; Colovic, Milena; Yuan, Zheliang; Merkens, Helen; Martin, Rainer E.; Benard, Francois; Schaffer, Paul; Britton, RobertJournal of the American Chemical Society (2017), 139 (10), 3595-3598CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A mild and selective photocatalytic C-H 18F-fluorination reaction has been developed that provides direct access to 18F-fluorinated amino acids. The biodistribution and uptake of three 18F-labeled leucine analogs via LAT1 mediated transport in several cancer cell lines is reported. Positron emission tomog. imaging of mice bearing PC3 (prostate) or U87 (glioma) xenografts using 5-[18F]-fluorohomoleucine showed high tumor uptake and excellent tumor visualization, highlighting the utility of this strategy for rapid tracer discovery for oncol.
- 29Limanto, J.; Shafiee, A.; Devine, P. N.; Upadhyay, V.; Desmond, R. A.; Foster, B. R.; Gauthier, D. R., Jr.; Reamer, R. A.; Volante, R. P. An efficient chemoenzymatic approach to (S)-γ-fluoroleucine ethyl ester. J. Org. Chem. 2005, 70, 2372– 2375, DOI: 10.1021/jo047918j[ACS Full Text], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1yisrs%253D&md5=1677d7e61b539f1ad4e19866ac7fdf92An Efficient Chemoenzymatic Approach to (S)-γ-Fluoroleucine Ethyl EsterLimanto, John; Shafiee, Ali; Devine, Paul N.; Upadhyay, Veena; Desmond, Richard A.; Foster, Bruce R.; Gauthier, Donald R., Jr.; Reamer, Robert A.; Volante, R. P.Journal of Organic Chemistry (2005), 70 (6), 2372-2375CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)This work describes the asym. synthesis of (S)-γ-fluoroleucine Et ester (I) from Ph2C:NCH2CO2Et starting material. The key reaction step involves a lipase-catalyzed dynamic ring-opening of 2-(3-butenyl)azlactone II (prepd. from Ph2C:NCH2CO2Et in 5 steps) with EtOH to give N-(pentenoyl)fluoroleucine ester III in 84% enantiomeric excess. Removal of the N-pentenoyl group from III with N,N'-dibromodimethylhydantoin (DBDMH) in the presence of TFA afforded I, which was isolated as its hydrogen sulfate salt in 75% yield and >97% enantiomeric excess.
- 30O’Shea, P. D.; Chen, C. Y.; Gauvreau, D.; Gosselin, F.; Hughes, G.; Nadeau, C.; Volante, R. P. A practical enantioselective synthesis of odanacatib, a potent Cathepsin K inhibitor, via triflate displacement of an α-trifluoromethylbenzyl triflate. J. Org. Chem. 2009, 74, 1605– 1610, DOI: 10.1021/jo8020314[ACS Full Text], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXlt1Sksw%253D%253D&md5=1edffc0a6a028f3e863effbe5a7c9639A Practical Enantioselective Synthesis of Odanacatib, a Potent Cathepsin K Inhibitor, via Triflate Displacement of an α-Trifluoromethylbenzyl TriflateO'Shea, Paul D.; Chen, Cheng-yi; Gauvreau, Danny; Gosselin, Francis; Hughes, Greg; Nadeau, Christian; Volante, Ralph P.Journal of Organic Chemistry (2009), 74 (4), 1605-1610CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)An enantioselective synthesis of the Cathepsin K inhibitor odanacatib (I, MK-0822) is described. The key step involves the novel stereospecific SN2 triflate displacement of a chiral α-trifluoromethylbenzyl triflate II with (S)-γ-fluoroleucine Et ester (III) to generate the required α-trifluoromethylbenzyl amino stereocenter. The triflate displacement is achieved in high yield (95%) and minimal loss of stereochem. The overall synthesis of I is completed in 6 steps in 61% overall yield.
- 31Wang, L.; Wei, J.; Wu, R.; Cheng, G.; Li, X.; Hu, J.; Hu, Y.; Sheng, R. The stability and reactivity of tri-, di-, and monofluoromethyl/methoxy/methylthio groups on arenes under acidic and basic conditions. Org. Chem. Front. 2017, 4, 214– 223, DOI: 10.1039/C6QO00674D[Crossref], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvVKqtbfK&md5=05878f761980875b1f59c72753825a4eThe stability and reactivity of tri-, di-, and monofluoromethyl/methoxy/methylthio groups on arenes under acidic and basic conditionsWang, Lingfei; Wei, Jun; Wu, Ranran; Cheng, Gang; Li, Xinjin; Hu, Jinbo; Hu, Yongzhou; Sheng, RongOrganic Chemistry Frontiers (2017), 4 (2), 214-223CODEN: OCFRA8; ISSN:2052-4129. (Royal Society of Chemistry)In this study, the stability and reactivity of tri-, di- and monofluoromethyl/methoxy/methylthio moiety groups on arenes under acidic or basic conditions was carefully investigated.
- 32Deng, H.; O’Hagan, D.; Schaffrath, C. Fluorometabolite biosynthesis and the fluorinase from Streptomyces cattleya. Nat. Prod. Rep. 2004, 21, 773– 784, DOI: 10.1039/b415087m[Crossref], [PubMed], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXmtVemug%253D%253D&md5=422f9ea144e9a98acd421b4f0df174b4Fluorometabolite biosynthesis and the fluorinase from Streptomyces cattleyaDeng, Hai; O'Hagan, David; Schaffrath, ChristophNatural Product Reports (2004), 21 (6), 773-784CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)A review. This review outlines the recent developments in uncovering the enzymes and intermediates involved in fluorometabolite biosynthesis in the bacterium Streptomyces cattleya. A particular emphasis is placed on the purifn. and characterization of the fluorinase, the C-F bond forming enzyme which initiates the biosynthesis. Nature has hardly developed a biochem. around fluorine, yet fluorinated orgs. are important com. entities, therefore a biotransformation from inorg. to org. fluorine is novel and of contemporary interest.
- 33Lee, S. T.; Cook, D.; Pfister, J. A.; Allen, J. G.; Colegate, S. M.; Riet-Correa, F.; Taylor, C. M. Monofluoroacetate-containing plants that are potentially toxic to livestock. J. Agric. Food Chem. 2014, 62, 7345– 7354, DOI: 10.1021/jf500563h[ACS Full Text], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmtVWhtbY%253D&md5=6368642dc811212d4ee11ac613a6a562Monofluoroacetate-Containing Plants That Are Potentially Toxic to LivestockLee, Stephen T.; Cook, Daniel; Pfister, James A.; Allen, Jeremy G.; Colegate, Steven M.; Riet-Correa, Franklin; Taylor, Charlotte M.Journal of Agricultural and Food Chemistry (2014), 62 (30), 7345-7354CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)A review. Many plants worldwide contain monofluoroacetate and cause sudden death in livestock. These plants are primarily found in the southern continents of Africa, Australia, and South America, where they neg. affect livestock prodn. This review highlights past and current research investigating the plants reported to contain monofluoroacetate and cause sudden death; the mode of action, clin. signs, and pathol. assocd. with poisoning by monofluoroacetate-contg. plants; chem. for the anal. of monofluoroacetate in plants; the coevolution of native flora and fauna in Western Australia with respect to monofluoroacetate-contg. plants; and methods to mitigate livestock losses caused by monofluoroacetate-contg. plants.
- 34Deng, H.; O’Hagan, D. The fluorinase, the chlorinase and the duf-62 enzymes. Curr. Opin. Chem. Biol. 2008, 12, 582– 592, DOI: 10.1016/j.cbpa.2008.06.036[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXht1KgsbbL&md5=b94069dc2015c2fb91f65b1a20c44cd6The fluorinase, the chlorinase and the duf-62 enzymesDeng, Hai; O'Hagan, DavidCurrent Opinion in Chemical Biology (2008), 12 (5), 582-592CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. The fluorinase from Streptomyces cattleya and chlorinase from Salinispora tropica have a commonality in that they mediate nucleophilic reactions of their resp. halide ions to the C-5' carbon of S-adenosyl-L-methionine (SAM). These enzyme reactions fall into the relatively small group of SN2 substitution reactions found in enzymol. These enzymes have some homol. to a larger class of proteins expressed by the duf-62 gene, of which around 200 representatives have been sequenced and deposited in databases. The duf-62 genes express a protein which mediates a hydrolytic cleavage of SAM to generate adenosine and L-methionine. Superficially this enzyme operates very similarly to the halogenases in that water/hydroxide replaces the halide ion. However structural examn. of the duf-62 gene product reveals a very different organization of the active site suggesting a novel mechanism for water activation.
- 35Champagne, P. A.; Pomarole, J.; Therien, M. E.; Benhassine, Y.; Beaulieu, S.; Legault, C. Y.; Paquin, J. F. Enabling nucleophilic substitution reactions of activated alkyl fluorides through hydrogen bonding. Org. Lett. 2013, 15, 2210– 2213, DOI: 10.1021/ol400765a[ACS Full Text], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXms1Wisrw%253D&md5=f3a55eb15516e129876c65a0b13b4061Enabling Nucleophilic Substitution Reactions of Activated Alkyl Fluorides through Hydrogen BondingChampagne, Pier Alexandre; Pomarole, Julien; Therien, Marie-Eve; Benhassine, Yasmine; Beaulieu, Samuel; Legault, Claude Y.; Paquin, Jean-FrancoisOrganic Letters (2013), 15 (9), 2210-2213CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)It was discovered that the presence of water as a cosolvent enables the reaction of activated alkyl fluorides for bimol. nucleophilic substitution reactions. DFT calcns. show that activation proceeds through stabilization of the transition structure by a stronger F···H2O interaction and diminishing C-F bond elongation, and not simple transition state electrostatic stabilization. Overall, the findings put forward a distinct strategy for C-F bond activation through H-bonding.
- 36O’Hagan, D.; Deng, H. Enzymatic fluorination and biotechnological developments of the fluorinase. Chem. Rev. 2015, 115, 634– 649, DOI: 10.1021/cr500209t[ACS Full Text], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1Wlu7zK&md5=316520768c80bde6f16f7248ef8f7734Enzymatic Fluorination and Biotechnological Developments of the FluorinaseO'Hagan, David; Deng, HaiChemical Reviews (Washington, DC, United States) (2015), 115 (2), 634-649CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review focusing on the biosynthesis fluorinated metabolites in nature, the enzymes involeved in fluorination and possible biotech. applications of these enzymes.
- 37Senn, H. M.; O’Hagan, D.; Thiel, W. Insight into enzymatic C–F bond formation from QM and QM/MM calculations. J. Am. Chem. Soc. 2005, 127, 13643– 13655, DOI: 10.1021/ja053875s[ACS Full Text], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXpvFSnurg%253D&md5=158d59340bfea62971bb86cb66d1570fInsight into Enzymatic C-F Bond Formation from QM and QM/MM CalculationsSenn, Hans Martin; O'Hagan, David; Thiel, WalterJournal of the American Chemical Society (2005), 127 (39), 13643-13655CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The C-F bond-forming step in the fluorinase, the only native fluorination enzyme characterized to date, has been studied. The enzyme catalyzes the reaction between S-adenosyl-L-methionine (SAM) and fluoride ions to form 5'-fluoro-5'-deoxyadenosine (5'-FDA) and L-methionine. To obtain an insight into the mechanism of this unusual enzymic reaction and to elucidate the role of the enzyme in catalysis, we have explored the conformational energetics of SAM and the intrinsic reactivity patterns of SAM and fluoride with DFT (BP86) and continuum solvent methods, before investigating the full enzymic system with combined DFT/CHARMM calcns. We find that the enzymic reaction follows an SN2 reaction mechanism, concurring with the intrinsic reactivity preferences in soln. The formation of sulfur ylides is thermodynamically strongly disfavored, and an alternative elimination-addn. mechanism involving the concerted anti-Markovnikov addn. of HF to an enol ether is energetically viable, but kinetically prohibitive. The SN2 activation energy is 92 (112) kJ mol-1 in soln., but only 53 (63) kJ mol-1 in the enzyme, and the reaction energy in the enzyme is -25 (-34) kJ mol-1 (values in parentheses are B3LYP single-point energies). The fluorinase thus lowers the barrier for C-F bond formation by 39 (49) kJ mol-1. A decompn. anal. shows that the major role of the enzyme is in the prepn. and positioning of the substrates.
- 38Zhu, X.; Robinson, D. A.; McEwan, A. R.; O’Hagan, D.; Naismith, J. H. Mechanism of enzymatic fluorination in Streptomyces cattleya. J. Am. Chem. Soc. 2007, 129, 14597– 14604, DOI: 10.1021/ja0731569[ACS Full Text], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1KisbfP&md5=b5155938982c64f203b4d81e1a1dfb9bMechanism of Enzymatic Fluorination in Streptomyces cattleyaZhu, Xiaofeng; Robinson, David A.; McEwan, Andrew R.; O'Hagan, David; Naismith, James H.Journal of the American Chemical Society (2007), 129 (47), 14597-14604CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Recently a fluorination enzyme was identified and isolated from Streptomyces cattleya, as the first committed step on the metabolic pathway to the fluorinated metabolites, fluoroacetate and 4-fluorothreonine. This enzyme, 5'-fluoro-5'-deoxy adenosine synthetase (FDAS), has been shown to catalyze C-F bond formation by nucleophilic attack of fluoride ion to S-adenosyl-L-methionine (SAM) with the concomitant displacement of L-methionine to generate 5'-fluoro-5'-deoxy adenosine (5'-FDA). Although the structures of FDAS bound to both SAM and products have been solved, the mol. mechanism remained to be elucidated. We now report site-directed mutagenesis studies, structural analyses, and isothermal calorimetry (ITC) expts. The data establish the key residues required for catalysis and the order of substrate binding. Fluoride ion is not readily distinguished from water by protein X-ray crystallog.; however, using chloride ion (also a substrate) with a mutant of low activity has enabled the halide ion to be located in nonproductive co-complexes with SAH and SAM. The kinetic data suggest the pos. charged sulfur of SAM is a key requirement in stabilizing the transition state. We propose a mol. mechanism for FDAS in which fluoride weakly assocs. with the enzyme exchanging two water mols. for protein ligation. The binding of SAM expels remaining water assocd. with fluoride ion and traps the ion in a pocket positioned to react with SAM, generating L-methionine and 5'-FDA. L-Methionine then dissocs. from the enzyme followed by 5'-FDA.
- 39Murphy, C. D.; O’Hagan, D.; Schaffrath, C. Identification of a PLP-dependent threonine transaldolase: A novel enzyme involved in 4-fluorothreonine biosynthesis in Streptomyces cattleya. Angew. Chem., Int. Ed. 2001, 40, 4479– 4481, DOI: 10.1002/1521-3773(20011203)40:23<4479::AID-ANIE4479>3.0.CO;2-1[Crossref], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXptlWhtbk%253D&md5=bf7ded2b1b7e78827d49625c641379e9Identification of a PLP-dependent threonine transaldolase: A novel enzyme involved in 4-fluorothreonine biosynthesis in Streptomyces cattleyaMurphy, Cormac D.; O'Hagan, David; Schaffrath, ChristophAngewandte Chemie, International Edition (2001), 40 (23), 4479-4481CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)The final enzyme on the biosynthetic pathway to 4-fluorothreonine is identified in Streptomyces cattleya. The enzyme catalyzes a pyridoxal phosphate-dependent "transaldose" reaction between L-threonine and fluoroacetaldehyde (PLP = pyridoxal 5'-phosphate). Unlike threonine aldolases, glycine is not a substrate for this new type of enzyme.
- 40Xu, X. H.; Yao, G. M.; Li, Y. M.; Lu, J. H.; Lin, C. J.; Wang, X.; Kong, C. H. 5-Fluorouracil derivatives from the sponge Phakellia fusca. J. Nat. Prod. 2003, 66, 285– 288, DOI: 10.1021/np020034f[ACS Full Text], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhtlCltA%253D%253D&md5=9f1432a7c640bd8b57437ed80ae814995-Fluorouracil Derivatives from the Sponge Phakellia fuscaXu, Xiao-Hua; Yao, Guang-Min; Li, Yan-Ming; Lu, Jian-Hua; Lin, Chang-Jiang; Wang, Xin; Kong, Chui-HuaJournal of Natural Products (2003), 66 (2), 285-288CODEN: JNPRDF; ISSN:0163-3864. (American Chemical Society)5-Fluorouracil derivs. were isolated from the marine sponge Phakellia fusca collected around the Yongxing Island of the Xisha Islands in the South Sea of China. Their structures were detd. on the basis of spectral anal. and X-ray diffraction.
- 41Goncharov, N. V.; Jenkins, R. O.; Radilov, A. S. Toxicology of fluoroacetate: a review, with possible directions for therapy research. J. Appl. Toxicol. 2006, 26, 148– 161, DOI: 10.1002/jat.1118[Crossref], [PubMed], [CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjtFerurs%253D&md5=d2e2b695c218a75c93b6a0c779e0bebbToxicology of fluoroacetate: a review, with possible directions for therapy researchGoncharov, Nikolay V.; Jenkins, Richard O.; Radilov, Andrey S.Journal of Applied Toxicology (2006), 26 (2), 148-161CODEN: JJATDK; ISSN:0260-437X. (John Wiley & Sons Ltd.)A review. Fluoroacetate (FA; CH2FCOOR) is highly toxic towards humans and other mammals through inhibition of the enzyme aconitase in the tricarboxylic acid cycle, caused by 'lethal synthesis' of an isomer of fluorocitrate (FC). FA is found in a range of plant species and their ingestion can cause the death of ruminant animals. Some fluorinated compds. used as anticancer agents, narcotic analgesics, pesticides or industrial chems. metabolize to FA as intermediate products. The chem. characteristics of FA and the clin. signs of intoxication warrant the re-evaluation of the toxic danger of FA and renewed efforts in the search for effective therapeutic means. Antidotal therapy for FA intoxication was aimed at preventing fluorocitrate synthesis and aconitase blockade in mitochondria, and at providing citrate outflow from this organelle. Despite a greatly improved understanding of the biochem. mechanism of FA toxicity, ethanol, if taken immediately after the poisoning, was the most acceptable antidote for the past 6 decades. This review deals with the clin. signs and physiol. and biochem. mechanisms of FA intoxication to provide an explanation of why, even after decades of investigation, has no effective therapy to FA intoxication been elaborated. An apparent lack of integrated toxicol. studies is viewed as a limiter of progress in this regard. Two principal ways of developing effective therapies for FA intoxication are considered. Firstly, competitive inhibition of FA interaction with CoA and of FC interaction with aconitase. Secondly, channeling the alternative metabolic pathways by orienting the fate of citrate via cytosolic aconitase, and by maintaining the flux of reducing equiv. into the TCA cycle via glutamate dehydrogenase.
- 42Kalmbach, E. R. ″Ten-Eighty,″ a war-produced rodenticide. Science 1945, 102, 232– 233, DOI: 10.1126/science.102.2644.232[Crossref], [PubMed], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3czotFyksg%253D%253D&md5=d64aff369b7492cde525d381dfa1bea9"TEN-EIGHTY," A WAR-PRODUCED RODENTICIDEKalmbach E RScience (New York, N.Y.) (1945), 102 (2644), 232-3 ISSN:0036-8075.There is no expanded citation for this reference.
- 43McCombie, H.; Saunders, B. C. Fluoroacetates and allied compounds. Nature 1946, 158, 382– 385, DOI: 10.1038/158382a0[Crossref], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaH2sXhtlOltg%253D%253D&md5=7c7d3a6b44d34cf35f49ca5d7da5354eFluoroacetates and allied compoundsMcCombie, H.; Saunders, B. C.Nature (London, United Kingdom) (1946), 158 (), 382-5CODEN: NATUAS; ISSN:0028-0836.Convulsant poisons with delayed action contg. the -CH2F group have been synthesized and investigated pharmacologically. The firm binding of the F renders chem. detection and decontamination difficult. A compd. of outstanding interest is 2-fluoroethyl fluoroacetate FCH2COOCH2CH2F (I) prepd. by the action of fluoroacetyl chloride (II) on 2-fluoroethyl alc. (III). A 10-min. exposure to 0.092 g. I/cu. m. killed 70% of the rabbits, guinea pigs, and rats tested and 0.05 g. I/cu. m. killed 50% of rabbits. About half as toxic are Me fluoroacetate (IV), b. 104° m. -35° and Et, Pr, and iso-Pr fluoroacetates, prepd. by heating the corresponding chloroacetates with KF in a rotating autoclave. IV usually produced convulsions 30-60 min. after exposure and death within a few hrs.; 0.1 g./cu. m. inhaled 10 min. killed 50% of the rabbits and guinea pigs (mice were somewhat more resistant), and 0.25 mg./kg. injected intravenously killed 50% of the rabbits. Claisen ester condensation of IV gave Me α γ-difluoroacetoacetate. Fluoroacetamide (V), prepd. from NH3 and IV, was useful for characterization purposes and as an intermediate in the prepn. of fluoroacetic acid (VI). V and VI were as toxic as IV on intravenous injection into rabbits. FCH2CN, b. 80° prepd. by distn. of V with phosphoric anhydride, was more toxic to rabbits than to smaller animals. Substituted amides prepd. included FCH2CONHMe, FCH2CON(NO)Me, FCH2CONH(CH2)2OH, FCH2CONH(CH2)2Cl, and FCH2CON(CH2CH2Cl)2. Na fluoroacetate (VII) was obtained by a new method from IV. II and fluoroacetyl fluoride were similar to IV in toxicity. Fluoroacetic anhydride, prepd. from VII and II, were rather more toxic by inhalation than IV. Et N-fluoroacetyl-glycine and cholesterol fluoroacetate were considerably less toxic than IV. III, b. 101° completely miscible with H2O, prepd. by heating ethylene chlorohydrin with KF in the rotating autoclave, was about as toxic as IV. I, III, and IV had extremely faint odors. Fluoroacetaldehyde, prepd. by oxidation of III with MnO2 and H2SO4, was a liquid which polymerized on standing and was similar to III in toxicity. FCH2CH2Cl obtained, by the action of SOCl2 on III, was nontoxic; it reacted with C6H5ONa to give Ph 2-fluoroethyl ether, considerably less toxic than IV. FCH2CH2Br (VIII) reacted with KSCN to give fluoroethyl thiocyanate, which on treatment with Cl-H2O gave fluoroethylsulfonyl chloride, nontoxic. Bis(2-fluoroethyl) sulfate, (FCH2CH2O)2SO2, proved useful as a fluoroethylating agent, e.g., in the reaction with β-naphthol in alk. soln. to form fluoroethyl naphthyl ether. 2-Chloroethyl fluoroacetate and 2-fluoroethyl chloroacetate (IX) were somewhat more toxic, while 2-fluoroethyl acetate and FCH2COSCH2CH2Cl were less toxic than IV. S,S'-Bis(2-fluoroethyl)-dithioglycol (X) FCH2CH2SCH2CH2SCH2CH2F, ("sesqui-fluoro-H") was nonvesicant and nontoxic, in contrast to S, S'-bis(2-chloroethyl)dithioglycol ("sesqui-H"). X was prepd. by the following reactions: VIII + NaSH → FCH2CH2SH → FCH2CH2SNa (XI); VIII + 2 XI → X. VIII reacted with trimethylamine, triethylamine, and pyridine to form (2-fluoroethyl)trimethylammonium bromide, (2-fluoroethyl)triethylammonium bromide, and (2-fluoroethyl)pyridinium bromide, resp., all not very toxic. Treatment of VIII with dimethylaniline gave 2-Fluoroethylglycine-HCl, prepd. by Fischer-Speier esterification of glycine with III, and 2-fluoroethylbetaine-HCl, prepd. by reaction of anhyd. trimethylamine and IX, had subcutaneous LD50 values of 10 and 45 mg./kg. for mice. Fluoroacetylsalicyclic acid (fluoroaspirin) caused initial stupor without convulsions in mice. Bis(2-fluoroethyl)fluorophosphonate, obtained by the action of POCl2F on III caused miosis, and at a concn. of 0.5 g./cu. m. (10 min. exposure) produced in 2 of 6 rats a remarkable state of hyperactivity followed by convulsions, coma, and death. Triethyllead fluoroacetate, FCH2COOPbEt3, had sternutatory and, upon injection, convulsant properties. No toxic action was shown by compds. devoid of the FCH2- group, i.e. Me α-fluoropropionate, Me α-fluoroisobutyrate, chloroacetyl fluoride, and Et fluoroformate.
- 44Lauble, H.; Kennedy, M. C.; Emptage, M. H.; Beinert, H.; Stout, C. D. The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 13699– 13703, DOI: 10.1073/pnas.93.24.13699[Crossref], [PubMed], [CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xnt1Gnsbo%253D&md5=ed8ef16ac9d474a434754199da264f02The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complexLauble, H.; Kennedy, M. C.; Emptage, M. H.; Beinert, H.; Stout, C. D.Proceedings of the National Academy of Sciences of the United States of America (1996), 93 (24), 13699-13703CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)It has been known for many years that fluoroacetate and fluorocitrate when metabolized are highly toxic, and that at least one effect of fluorocitrate is to inactivate aconitase. Here, the authors present evidence supporting the hypothesis that the (-)-erythro diastereomer of 2-fluorocitrate acts as a mechanism-based inhibitor of aconitase by 1st being converted to fluoro-cis-aconitate, followed by the addn. of hydroxide and with loss of fluoride to form 4-hydroxy-trans-aconitate (HTn), which binds very tightly, but not covalently, to the enzyme. The formation of HTn by these reactions is in accord with the working model for the enzyme mechanism. That HTn is the product of fluorocitrate inhibition is supported by the crystal structure of the enzyme-inhibitor complex at 2.05-Å resoln., release of fluoride stoichiometric with total enzyme when (-)-erythro-2-fluorocitrate is added, HPLC anal. of the product, slow displacement of HTn by 106-fold excess of isocitrate, and previously published Moessbauer expts. When (+)-erythro-2-fluorocitrate is added to aconitase, the release of fluoride is stoichiometric with total substrate added, and HPLC anal. of the products indicates the formation of oxalosuccinate, and its deriv., α-ketoglutarate. This is consistent with the proposed mechanism, as is the formation of HTn from (-)-erythro-2-fluorocitrate. The structure of the inhibited complex reveals that HTn binds like the inhibitor, trans-aconitate, while providing all the interactions of the natural substrate, isocitrate. The structure exhibits 4 H-bonds <2.7 Å in length involving HTn, H2O bound to the [4Fe-4S] cluster, Asp-165, and His-167, as well as low temp. factors for these moieties, consistent with the obsd. very tight binding of the inhibitor.
- 45Hamel, J. D.; Paquin, J. F. Activation of C–F bonds α to C–C multiple bonds. Chem. Commun. (Cambridge, U. K.) 2018, 54, 10224– 10239, DOI: 10.1039/C8CC05108A[Crossref], [PubMed], [CAS], Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsFCktbfN&md5=4c6e994215ac89974c861999f74f7b80Activation of C-F bonds α to C-C multiple bondsHamel, Jean-Denys; Paquin, Jean-FrancoisChemical Communications (Cambridge, United Kingdom) (2018), 54 (73), 10224-10239CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A review. C-F bond activation has drawn a lot of attention in the past, and the case of C(sp3)-F bonds is particularly interesting as those are the strongest single bonds carbon makes with any other element. This Feature Article aims at highlighting the authors' work and that of others on the successful approaches that led to the activation of such C-F bonds at benzylic, allylic, propargylic and allenylic positions, for both mono- and polyfluorides. The authors now hope that this will set ground for discussions, contribute to fostering new ideas, and thus help in pushing current frontiers further.
- 46Sherley, M. The traditional categories of fluoroacetate poisoning signs and symptoms belie substantial underlying similarities. Toxicol. Lett. 2004, 151, 399– 406, DOI: 10.1016/j.toxlet.2004.03.013[Crossref], [PubMed], [CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXlslyitLw%253D&md5=17bea1d92ea6abe9db66e73d61e04d97The traditional categories of fluoroacetate poisoning signs and symptoms belie substantial underlying similaritiesSherley, MirandaToxicology Letters (2004), 151 (3), 399-406CODEN: TOLED5; ISSN:0378-4274. (Elsevier Ireland Ltd.)A review. Sodium monofluoroacetate (Compd. 1080) has been widely used around the world as a vertebrate pest control agent. Following ingestion of 1080 there is a latent period, during which the compd. is metabolized into a toxic form, before the onset of symptoms. The timing of this period varies significantly between species as does the median LD. Traditionally different species have also been classified into groups depending on the primary organ system involved in 1080 toxicosis (cardiac, nervous, or mixed signs/symptoms). However, general acceptance of this method of classification has obscured the fact that several signs of fluoroacetate poisoning are common to most vertebrate species. This paper reviews 5 decades of literature on the signs/symptoms of fluoroacetate poisoning in vertebrates and concludes that there is little justification for the division of animals poisoned by fluoroacetate into symptomatic groups.
- 47Proudfoot, A. T.; Bradberry, S. M.; Vale, J. A. Sodium fluoroacetate poisoning. Toxicol. Rev. 2006, 25, 213– 219, DOI: 10.2165/00139709-200625040-00002[Crossref], [PubMed], [CAS], Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjs1Gksrs%253D&md5=7229d74004657a0ba412497dc809e2edSodium fluoroacetate poisoningProudfoot, Alex T.; Bradberry, Sally M.; Vale, J. AllisterToxicological Reviews (2006), 25 (4), 213-219CODEN: TROEB5; ISSN:1176-2551. (Adis International Ltd.)A review. Sodium fluoroacetate was introduced as a rodenticide in the US in 1946. However, its considerable efficacy against target species is offset by comparable toxicity to other mammals and, to a lesser extent, birds and its use as a general rodenticide was therefore severely curtailed by 1990. Currently, sodium fluoroacetate is licensed in the US for use against coyotes, which prey on sheep and goats, and in Australia and New Zealand to kill unwanted introduced species. The extreme toxicity of fluoroacetate to mammals and insects stems from its similarity to acetate, which has a pivotal role in cellular metab. Fluoroacetate combines with CoA (CoA-SH) to form fluoroacetyl CoA, which can substitute for acetyl CoA in the tricarboxylic acid cycle and reacts with citrate synthase to produce fluorocitrate, a metabolite of which then binds very tightly to aconitase thereby halting the cycle. Many of the features of fluoroacetate poisoning are, therefore, largely direct and indirect consequences of impaired oxidative metab. Energy prodn. is reduced and intermediates of the tricarboxylic acid cycle subsequent to citrate are depleted. Among these is oxoglutarate, a precursor of glutamate, which is not only an excitatory neurotransmitter in the CNS but is also required for efficient removal of ammonia via the urea cycle. Increased ammonia concns. may contribute to the incidence of seizures. Glutamate is also required for glutamine synthesis and glutamine depletion has been obsd. in the brain of fluoroacetate-poisoned rodents. Reduced cellular oxidative metab. contributes to a lactic acidosis. Inability to oxidize fatty acids via the tricarboxylic acid cycle leads to ketone body accumulation and worsening acidosis. ATP (ATP) depletion results in inhibition of high energy-consuming reactions such as gluconeogenesis. Fluoroacetate poisoning is assocd. with citrate accumulation in several tissues, including the brain. Fluoride liberated from fluoroacetate, citrate and fluorocitrate are calcium chelators and there are both animal and clin. data to support hypocalcemia as a mechanism of fluoroacetate toxicity. However, the available evidence suggests the fluoride component does not contribute. Acute poisoning with sodium fluoroacetate is uncommon. Ingestion is the major route by which poisoning occurs. Nausea, vomiting and abdominal pain are common within 1 h of ingestion. Sweating, apprehension, confusion and agitation follow. Both supraventricular and ventricular arrhythmias have been reported and nonspecific ST- and T-wave changes are common, the QTc may be prolonged and hypotension may develop. Seizures are the main neurol. feature. Coma may persist for several days. Although several possible antidotes have been investigated, they are of unproven value in humans. The immediate, and probably only, management of fluoroacetate poisoning is therefore supportive, including the correction of hypocalcemia.
- 48Kirsten, E.; Sharma, M. L.; Kun, E. Molecular toxicology of (−)-erythro-fluorocitrate: selective inhibition of citrate transport in mitochondria and the binding of fluorocitrate to mitochondrial proteins. Mol. Pharmacol. 1978, 14, 172– 184[PubMed], [CAS], Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXhsVKqs7c%253D&md5=a99fce7d622718212edbb608d9c716deMolecular toxicology of (-)-erythro-fluorocitrate: selective inhibition of citrate transport in mitochondria and the binding of fluorocitrate to mitochondrial proteinsKirsten, Eva; Sharma, Manohar L.; Kun, ErnestMolecular Pharmacology (1978), 14 (1), 172-84CODEN: MOPMA3; ISSN:0026-895X.The rate of entry of oxidizable carboxylic acids into the mitoplast compartment of lysosome- and microsome-free mitochondria was detd. by monitoring substrate-dependent incorporation of 32P-labeled orthophosphate into ADP to form ATP [56-65-5]. The maximal rate of ATP synthesis that was dependent on tricarboxylic acids required the presence of (-)-erythro-fluoromalate [34917-27-6], replacing the physiol. activator L-malate. Previous incubation of mitochondria for 5-15 min (at 25-30°) with 50 pmols of (-)-erythro-fluorocitrate [34917-26-5]/mg mitochondrial protein irreversibly and selectively inhibited citrate [77-92-9] supported ATP synthesis. Previous incubation of mitochondria for 15 min with quantities of (-)-erythro-fluorocitrate that inhibited citrate-dependent ATP synthesis also inhibited mitochondrial citrate-dependent fatty acid synthesis. The utilization of added citrate for fatty acid biosynthesis by the cytoplasmic system was not affected by fluorocitrate. Incubation of inner membrane vesicles with 3,4,5,6-14C-labeled (-)-erythro-fluorocitrate resulted in the covalent binding of fluorocitrate to protein components of the membrane. The bond between fluorocitric acid and protein was specifically cleaved by incubation with 0.4 M neutral hydroxylamine, but not by 1 mM o-phenanthroline. The hydroxamic acid of fluorocitrate, which was formed by treatment of protein-bound fluorocitrate with hydroxylamine, was detected by high-voltage electrophoresis and thin-layer chromatog. Binding of fluorocitrate to inner membrane vesicles dissolved in 4 M guanidine-HCl was prevented by previous incubation of membrane proteins with o-{[3-(hydroxymercuri)-2-methoxypropyl]carbamoyl}phenoxyacetic acid Na salt. The covalent binding of (-)-erythro-fluorocitrate to membrane proteins was the result of an enzymic process that was activated by Mn2+. Oxidizable carboxylic acids did not form covalently bound adducts with inner membrane proteins. It was concluded that (-)-erythro-fluorocitrate specifically inhibited citrate transport by covalent binding to 2 protein fractions assocd. with the mitoplast of liver, kidney, heart, and brain tissue.
- 49Menon, K. I.; Feldwick, M. G.; Noakes, P. S.; Mead, R. J. The mode of toxic action of the pesticide gliftor: the metabolism of 1,3-difluoroacetone to (−)-erythro-fluorocitrate. J. Biochem. Mol. Toxicol. 2001, 15, 47– 54, DOI: 10.1002/1099-0461(2001)15:1<47::AID-JBT6>3.0.CO;2-E[Crossref], [PubMed], [CAS], Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXotFWltA%253D%253D&md5=f768c20bacfd23f6a5cfdec35db822d0The mode of toxic action of the pesticide Gliftor: the metabolism of 1,3-difluoroacetone to (-)-erythro-fluorocitrateMenon, K. I.; Feldwick, M. G.; Noakes, P. S.; Mead, R. J.Journal of Biochemical and Molecular Toxicology (2001), 15 (1), 47-54CODEN: JBMTFQ; ISSN:1095-6670. (John Wiley & Sons, Inc.)The biochem. toxicol. of 1,3-difluoroacetone, a known metabolite of the major ingredient of the pesticide Gliftor (1,3-difluoro-2-propanol), was investigated in vivo and in vitro. Rat kidney homogenates supplemented with CoA, ATP, oxaloacetate, and Mg2+ converted 1,3-difluoroacetone to (-)-erythro-fluorocitrate in vitro. Administration of 1,3-difluoroacetone (100 mg kg-1 body wt.) to rats in vivo resulted in (-)-erythro-fluorocitrate synthesis in the kidney, which was preceded by an elevation in fluoride levels and followed by citrate accumulation. Animals dosed with 1,3-difluoroacetone did not display the 2-3 h lag phase in either (-)-erythro-fluorocitrate synthesis or in citrate and fluoride accumulation characteristic of animals dosed with 1,3-difluoro-2-propanol. We demonstrate that the conversion of 1,3-difluoro-2-propanol to 1,3-difluoroacetone by an NAD+-dependent oxidn. is the rate-limiting step in the synthesis of the toxic product, (-)-erythro-fluorocitrate from 1,3-difluoro-2-propanol. Prior administration of 4-methylpyrazole (90 mg kg-1 body wt.) was shown to prevent the conversion of 1,3-difluoro-2-propanol (100 mg kg-1 body wt.) to (-)-erythro-fluorocitrate in vivo and to eliminate the fluoride and citrate elevations seen in 1,3-difluoro-2-propanol-intoxicated animals. However, administration of 4-methylpyrazole (90 mg kg-1 body wt.) to rats 2 h prior to 1,3-difluoroacetone (100 mg kg-1 body wt.) was ineffective in preventing (-)-erythro-fluorocitrate synthesis and did not diminish fluoride or citrate accumulation in vivo. We conclude that the prophylactic and antidotal properties of 4-methylpyrazole seen in animals treated with 1,3-difluoro-2-propanol derive from its capacity to inhibit the NAD+-dependent oxidn. responsible for converting 1,3-difluoro-2-propanol to 1,3-difluoroacetone in the committed step of the toxic pathway.
- 50Feldwick, M. G.; Noakes, P. S.; Prause, U.; Mead, R. J.; Kostyniak, P. J. The biochemical toxicology of 1,3-difluoro-2-propanol, the major ingredient of the pesticide gliftor: the potential of 4-methylpyrazole as an antidote. J. Biochem. Mol. Toxicol. 1998, 12, 41– 52, DOI: 10.1002/(SICI)1099-0461(1998)12:1<41::AID-JBT6>3.0.CO;2-P[Crossref], [PubMed], [CAS], Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXnsl2ktLc%253D&md5=f87b7ff30d51b11fe12ebf72c023e82fThe biochemical toxicology of 1,3-difluoro-2-propanol, the major ingredient of the pesticide gliftor: the potential of 4-methylpyrazole as an antidoteFeldwick, M. G.; Noakes, P. S.; Prause, U.; Mead, R. J.; Kostyniak, P. J.Journal of Biochemical and Molecular Toxicology (1998), 12 (1), 41-52CODEN: JBMTFQ; ISSN:1095-6670. (John Wiley & Sons, Inc.)Administration to rats of 1,3-difluoro-2-propanol (100 mg kg-1 body wt.), the major ingredient of the pesticide gliftor, resulted in accumulation of citrate in the kidney after a 3 h lag phase. 1,3-Difluoro-2-propanol was found to be metabolized to 1,3-difluoroacetone and ultimately to the aconitate hydratase inhibitor (-)-erythrofluorocitrate and free fluoride. The conversion of 1,3-difluoro-2-propanol to 1,3-difluoroacetone was found to be catalyzed by an NAD+-dependent alc. dehydrogenase, while the defluorination was attributed to microsomal monooxygenase activity induced by phenobarbitone and inhibited by piperonyl butoxide. 4-Methylpyrazole was found to inhibit both of these processes in vitro and when administered (90 mg kg-1 body wt.) to rats, 2 h prior to 1,3-difluoro-2-propanol, eliminated signs of poisoning, prevented (-)-erythrofluorocitrate synthesis, and markedly decreased citrate and fluoride accumulation in vivo. 4-Methylpyrazole also appeared to diminish (-)-erythrofluorocitrate synthesis from fluoroacetate in vivo, and this was attributed to its capacity to inhibit malate dehydrogenase activity. The antidotal potential of 4-methylpyrazole and the potential for 1,3-difluoro-2-propanol to replace fluoroacetate (compd. 1080) as a vertebrate pesticide is discussed.
- 51Leisch, H.; Morley, K.; Lau, P. C. Baeyer-Villiger monooxygenases: more than just green chemistry. Chem. Rev. 2011, 111, 4165– 4222, DOI: 10.1021/cr1003437[ACS Full Text], [CAS], Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXls1elurw%253D&md5=4414d1675ebfeb634c52db50d1625a3dBaeyer-Villiger Monooxygenases: More Than Just Green ChemistryLeisch, Hannes; Morley, Krista; Lau, Peter C. K.Chemical Reviews (Washington, DC, United States) (2011), 111 (7), 4165-4222CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)This review focuses on the monooxygenase-catalyzed Baeyer-Villiger oxidn. of linear or cyclic ketones as a green chem. tool to address environmental sustainability, a system to study its mol. diversity and catalytic mechanism, industrial scale bioprocess development, and protein engineering to evolve new biotechnol. applications.
- 52Fiorentini, F.; Romero, E.; Fraaije, M. W.; Faber, K.; Hall, M.; Mattevi, A. Baeyer-Villiger monooxygenase FMO5 as entry point in drug metabolism. ACS Chem. Biol. 2017, 12, 2379– 2387, DOI: 10.1021/acschembio.7b00470[ACS Full Text], [CAS], Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1yisrfL&md5=f8c5f7d9f23f28fdda7e5de91bcb5ef2Baeyer-Villiger Monooxygenase FMO5 as Entry Point in Drug MetabolismFiorentini, Filippo; Romero, Elvira; Fraaije, Marco W.; Faber, Kurt; Hall, Melanie; Mattevi, AndreaACS Chemical Biology (2017), 12 (9), 2379-2387CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Flavin-contg. monooxygenases (FMOs) are emerging as effective players in oxidative drug metab. Until recently, the functions of the five human FMO isoforms were mostly linked to their capability of oxygenating mols. contg. soft N- and S-nucleophiles. However, the human FMO isoform 5 was recently shown to feature an atypical activity as Baeyer-Villiger monooxygenase. With the aim of evaluating such an alternative entry point in the metab. of active pharmaceutical ingredients, the authors selected and tested drug mols. bearing a carbonyl group on an aliph. chain. Nabumetone and pentoxifylline, two widely used pharmaceuticals, were thereby demonstrated to be efficiently oxidized in vitro by FMO5 to the corresponding acetate esters with high selectivity. The proposed pathways explain the formation of a predominant plasma metabolite of pentoxifylline as well as the crucial transformation of the pro-drug nabumetone into the pharmacol. active compd. Using the recombinant enzyme, the ester derivs. of both drugs were obtained in milligram amts., purified, and fully characterized. This protocol can potentially be extended to other FMO5 candidate substrates as it represents an effective and robust bench-ready platform applicable to API screening and metabolite synthesis.
- 53Fiorentini, F.; Geier, M.; Binda, C.; Winkler, M.; Faber, K.; Hall, M.; Mattevi, A. Biocatalytic characterization of human FMO5: Unearthing Baeyer-Villiger reactions in humans. ACS Chem. Biol. 2016, 11, 1039– 1048, DOI: 10.1021/acschembio.5b01016[ACS Full Text], [CAS], Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XoslymtA%253D%253D&md5=2d328642497c76a7c6789b0e70f1ed7fBiocatalytic Characterization of Human FMO5: Unearthing Baeyer-Villiger Reactions in HumansFiorentini, Filippo; Geier, Martina; Binda, Claudia; Winkler, Margit; Faber, Kurt; Hall, Melanie; Mattevi, AndreaACS Chemical Biology (2016), 11 (4), 1039-1048CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Flavin-contg. mono-oxygenases are known as potent drug-metabolizing enzymes, providing complementary functions to the well-investigated cytochrome P 450 mono-oxygenases. While human FMO isoforms are typically involved in the oxidn. of soft nucleophiles, the biocatalytic activity of human FMO5 (along its physiol. role) has long remained unexplored. In this study, we demonstrate the atypical in vitro activity of human FMO5 as a Baeyer-Villiger mono-oxygenase on a broad range of substrates, revealing the first example to date of a human protein catalyzing such reactions. The isolated and purified protein was active on diverse carbonyl compds., whereas soft nucleophiles were mostly non- or poorly reactive. The absence of the typical characteristic sequence motifs sets human FMO5 apart from all characterized Baeyer-Villiger mono-oxygenases so far. These findings open new perspectives in human oxidative metab.
- 54Kitazume, T.; Kataoka, J. Study on the effect of di- and trifluoromethyl groups on the Baeyer-Villiger reaction. J. Fluorine Chem. 1996, 80, 157– 158, DOI: 10.1016/S0022-1139(96)03500-2[Crossref], [CAS], Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXitVCgsA%253D%253D&md5=1f163743c0fa284d619a6e788312f67fStudy on the effect of di- and trifluoromethyl groups on the Baeyer-Villiger reactionKitazume, Tomoya; Kataoka, JunichiJournal of Fluorine Chemistry (1996), 80 (2), 157-158CODEN: JFLCAR; ISSN:0022-1139. (Elsevier)Case studies of the Baeyer-Villiger reaction applied to di- and trifluoromethyl ketone derivs. and the effect of the fluoromethyl groups on the Baeyer-Villiger reaction, are described.
- 55Kobayashi, S.; Tanaka, H.; Amii, H.; Uneyama, K. A new finding in selective Baeyer-Villiger oxidation of α-fluorinated ketones; a new and practical route for the synthesis of α-fluorinated esters. Tetrahedron 2003, 59, 1547– 1552, DOI: 10.1016/S0040-4020(03)00047-4[Crossref], [CAS], Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhtFCkt70%253D&md5=59ba46de3d9a7cdccdb389013f7bbe45A new finding in selective Baeyer-Villiger oxidation of α-fluorinated ketones; a new and practical route for the synthesis of α-fluorinated estersKobayashi, Satoru; Tanaka, Hiroaki; Amii, Hideki; Uneyama, KenjiTetrahedron (2003), 59 (9), 1547-1552CODEN: TETRAB; ISSN:0040-4020. (Elsevier Science Ltd.)α-Fluorinated esters were effectively prepd. by the Baeyer-Villiger oxidn. of α-fluorinated ketones with m-chloroperbenzoic acid (m-CPBA) under mild conditions. The yield of the esters was influenced by the choice of solvent, base, and substituent on the aryl group of the ketones. 4-Methoxyphenyl substituted fluoro-ketones were oxidized almost quant. with m-CPBA within 10 min to 12 h at room temp. using 1,1,1,3,3,3-hexafluoro-2-propanol as a cosolvent with CH2Cl2 (1:1, vol./vol.) and aq. buffer (KH2PO4-NaOH, pH 7.6) as an additive base. The oxidn. reaction rates of α-fluorinated ketones were higher than those of the corresponding non-fluorinated ketones. The fluorine atom at α-position of fluoromethyl aryl ketones enhanced the reactivity in the Baeyer-Villiger oxidn.
- 56Fischer, R. T.; Trzaskos, J. M.; Magolda, R. L.; Ko, S. S.; Brosz, C. S.; Larsen, B. Lanosterol 14α-methyl demethylase. Isolation and characterization of the third metabolically generated oxidative demethylation intermediate. J. Biol. Chem. 1991, 266, 6124– 6132[PubMed], [CAS], Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXktVequr0%253D&md5=9ffe09b0aa9c75e31bc57062ee2120e8Lanosterol 14α-methyl demethylase. Isolation and characterization of the third metabolically generated oxidative demethylation intermediateFischer, Robert T.; Trzaskos, James M.; Magolda, Ronald L.; Ko, Soo S.; Brosz, Christian S.; Larsen, BarbaraJournal of Biological Chemistry (1991), 266 (10), 6124-32CODEN: JBCHA3; ISSN:0021-9258.Conditions were identified which permit metabolic formation of the 3rd oxidized intermediate in the lanosterol 14α-Me demethylase reaction cascade. Metab. of either the immediate precursor substrate, 3β-hydroxylanost-8-en-32-al, or lanost-8-ene-3β,32-diol under mixed function oxidase conditions afforded formation of the intermediate. It must be emphasized that the intermediate could only be detected if sapon. procedures were omitted during sterol isolation. Comparative chem. and biochem. studies of the isolated metabolite with 3β,15α-dihydroxylanost-8-en-32-al revealed that the metabolite was not the 15α-hydroxylanosterol aldehyde, a putative demethylase intermediate. The metabolite was efficiently converted to the demethylated Δ8,14-diene sterol in the absence of O2 or NADPH, thus supporting its identity as the final oxidized intermediate in the lanosterol 14α-Me demethylase cascade. 1H NMR anal. showed a proton resonance at 7.86 ppm consistent with a formyloxy proton. Mass spectral and IR anal. of the metabolite clearly established O insertion into the immediate precursor substrate, 3β-hydroxylanost-8-en-32-al. Collectively, the biochem. and chem. characteristics of the metabolite supported a structural assignment for the metabolite as 14α-formyloxylanost-8-en-3β-ol.
- 57Vaz, A. D.; Pernecky, S. J.; Raner, G. M.; Coon, M. J. Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 4644– 4648, DOI: 10.1073/pnas.93.10.4644[Crossref], [PubMed], [CAS], Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XjtVKlsr8%253D&md5=1b5010f98101cdc729d53f277671f10ePeroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4Vaz, Alfin D. N.; Pernecky, Steven J.; Raner, Gregory M.; Coon, Minor J.Proceedings of the National Academy of Sciences of the United States of America (1996), 93 (10), 4644-4648CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Among biol. catalysts, cytochrome P 450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chem. reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several labs. points to a hypervalent iron-oxenoid species in P 450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P 450 aromatase and iron-peroxo species is involved. We have shown more recently that purified liver microsomal P 450 cytochromes, including phenobarbital-induced P 450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P 450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (Δ2-27)] makes use of evidence from other labs. that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidn., hydrogen peroxide prodn., and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidn., cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were detd. with P450s 2B4, 2B4 (Δ2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenyl-ethanol (2-fold). In sharp contrast, the deformylation of cyclohexane by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H2O2, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P 450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.
- 58Isin, E. M.; Guengerich, F. P. Complex reactions catalyzed by cytochrome P450 enzymes. Biochim. Biophys. Acta, Gen. Subj. 2007, 1770, 314– 329, DOI: 10.1016/j.bbagen.2006.07.003[Crossref], [PubMed], [CAS], Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXpvVyqsQ%253D%253D&md5=05b988e6536ea1e8725799d3cf395005Complex reactions catalyzed by cytochrome P 450 enzymesIsin, Emre M.; Guengerich, F. PeterBiochimica et Biophysica Acta, General Subjects (2007), 1770 (3), 314-329CODEN: BBGSB3; ISSN:0304-4165. (Elsevier Ltd.)A review. Cytochrome P 450 (P 450) isoforms are some of the most versatile redox proteins known. The basic P 450 reactions include C-hydroxylation, heteroatom oxygenation, heteroatom release (dealkylation), and epoxide formation. Mechanistic explanations for these reactions have been advanced. A no. of more complex P 450 reactions also occur, and these can be understood largely in the context of the basic chem. mechanisms and subsequent rearrangements. The list discussed here updates a 2001 review and includes Cl oxygenation, arom. dehalogenation, formation of diindole products, dimer formation via Diels-Alder reactions of products, ring coupling and also ring formation, reductive activation (e.g., aristolochic acid), ring contraction (piperidine nitroxide radical), oxidn. of troglitazone, cleavage of amino oxazoles and a 1,2,4-oxadiazole ring, bioactivation of a dihydrobenzoxathiin, and oxidative aryl migration.
- 59Guengerich, F. P.; Yoshimoto, F. K. Formation and cleavage of C–C bonds by enzymatic oxidation-reduction reactions. Chem. Rev. 2018, 118, 6573– 6655, DOI: 10.1021/acs.chemrev.8b00031[ACS Full Text], [CAS], Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFOru7fJ&md5=53975521b83d7596b867cf27adbd59b7Formation and Cleavage of C-C Bonds by Enzymatic Oxidation-Reduction ReactionsGuengerich, F. Peter; Yoshimoto, Francis K.Chemical Reviews (Washington, DC, United States) (2018), 118 (14), 6573-6655CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Many oxidn.-redn. (redox) enzymes, particularly oxygenases, have roles in reactions that make and break C-C bonds. The list includes cytochrome P 450 and other heme-based monooxygenases, heme-based dioxygenases, non-heme iron mono- and dioxygenases, flavoproteins, radical S-adenosylmethionine enzymes, copper enzymes, and peroxidases. Reactions involve steroids, intermediary metab., secondary natural products, drugs, and industrial and agricultural chems. Many C-C bonds are formed via either (i) coupling of diradicals or (ii) generation of unstable products that rearrange. C-C cleavage reactions involve several themes: (i) rearrangement of unstable oxidized products produced by the enzymes, (ii) oxidn. and collapse of radicals or cations via rearrangement, (iii) oxygenation to yield products that are readily hydrolyzed by other enzymes, and (iv) activation of O2 in systems in which the binding of a substrate facilitates O2 activation. Many of the enzymes involve metals, but of these iron is clearly predominant.
- 60Cox, C. D.; Coleman, P. J.; Breslin, M. J.; Whitman, D. B.; Garbaccio, R. M.; Fraley, M. E.; Buser, C. A.; Walsh, E. S.; Hamilton, K.; Schaber, M. D.; Lobell, R. B.; Tao, W.; Davide, J. P.; Diehl, R. E.; Abrams, M. T.; South, V. J.; Huber, H. E.; Torrent, M.; Prueksaritanont, T.; Li, C.; Slaughter, D. E.; Mahan, E.; Fernandez-Metzler, C.; Yan, Y.; Kuo, L. C.; Kohl, N. E.; Hartman, G. D. Kinesin spindle protein (KSP) inhibitors. 9. Discovery of (2S)-4-(2,5-difluorophenyl)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide (MK-0731) for the treatment of taxane-refractory cancer. J. Med. Chem. 2008, 51, 4239– 4252, DOI: 10.1021/jm800386y[ACS Full Text], [CAS], Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnsl2rs7s%253D&md5=283d7227309cbe634f18a640c8085090Kinesin Spindle Protein (KSP) Inhibitors. 9. Discovery of (2S)-4-(2,5-Difluorophenyl)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide (MK-0731) for the Treatment of Taxane-Refractory CancerCox, Christopher D.; Coleman, Paul J.; Breslin, Michael J.; Whitman, David B.; Garbaccio, Robert M.; Fraley, Mark E.; Buser, Carolyn A.; Walsh, Eileen S.; Hamilton, Kelly; Schaber, Michael D.; Lobell, Robert B.; Tao, Weikang; Davide, Joseph P.; Diehl, Ronald E.; Abrams, Marc T.; South, Vicki J.; Huber, Hans E.; Torrent, Maricel; Prueksaritanont, Thomayant; Li, Chunze; Slaughter, Donald E.; Mahan, Elizabeth; Fernandez-Metzler, Carmen; Yan, Youwei; Kuo, Lawrence C.; Kohl, Nancy E.; Hartman, George D.Journal of Medicinal Chemistry (2008), 51 (14), 4239-4252CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Inhibition of kinesin spindle protein (KSP) is a novel mechanism for treatment of cancer with the potential to overcome limitations assocd. with currently employed cytotoxic agents. Herein, we describe a C2-hydroxymethyl dihydropyrrole KSP inhibitor (11) that circumvents hERG channel binding and poor in vivo potency, issues that limited earlier compds. from our program. However, introduction of the C2-hydroxymethyl group caused 11 to be a substrate for cellular efflux by P-glycoprotein (Pgp). Utilizing knowledge garnered from previous KSP inhibitors, we found that β-fluorination modulated the pKa of the piperidine nitrogen and reduced Pgp efflux, but the resulting compd. (14) generated a toxic metabolite in vivo. Incorporation of fluorine in a strategic, metabolically benign position by synthesis of an N-methyl-3-fluoro-4-(aminomethyl)piperidine urea led to compd. 30 that has an optimal in vitro and metabolic profile. Compd. 30 (MK-0731) was recently studied in a phase I clin. trial in patients with taxane-refractory solid tumors.
- 61Eichhold, T. H.; Hookfin, E. B.; Taiwo, Y. O.; De, B.; Wehmeyer, K. R. Isolation and quantification of fluoroacetate in rat tissues, following dosing of Z-Phe-Ala-CH2-F, a peptidyl fluoromethyl ketone protease inhibitor. J. Pharm. Biomed. Anal. 1997, 16, 459– 467, DOI: 10.1016/S0731-7085(97)00102-7[Crossref], [PubMed], [CAS], Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXnvFWqsL8%253D&md5=ea69dcdcaff325ed19d40e94c6f96760Isolation and quantification of fluoroacetate in rat tissues, following dosing of Z-Phe-Ala-CH2-F, a peptidyl fluoromethyl ketone protease inhibitorEichhold, Thomas H.; Hookfin, Erin B.; Taiwo, Yetunde O.; De, Biswanath; Wehmeyer, Kenneth R.Journal of Pharmaceutical and Biomedical Analysis (1997), 16 (3), 459-467CODEN: JPBADA; ISSN:0731-7085. (Elsevier Science B.V.)Peptidyl fluoromethyl ketones (PFMK) are irreversible inhibitors of cathepsin B, a cysteine proteinase thought to be involved in the degrdn. of cartilage. It has been speculated that PFMK inhibitors may metabolize in rodents to form fluoroacetate (FAC), an extremely toxic poison. A highly selective and sensitive sepn. and detection scheme was developed to measure trace levels of FAC in rat tissues following PFMK dosing. The procedure consisted of extg. FAC from tissue and spiking the ext. with [18O]2-fluoroacetate (18O-FAC) as an internal std. FAC and 18O-FAC were further isolated from matrix components using ion-exchange, solid-phase extn. The pentafluorobenzyl esters of FAC and 18O-FAC were formed to facilitate the chromatog. sepn. Two-dimensional gas chromatog. coupled with selected-ion-monitoring detection provided the final measurement. The assay had a limit of detection of 2 ng FAC per g tissue, and was capable of accurately quantitating as little as 10 ng FAC per g tissue with a S/N ratio of 40:1. Linearity was established over two orders of magnitude, from 2-500 ng ml-1, with 5 μl injected on-column. The method was used to demonstrate that FAC was formed in rats following dosing with Z-Phe-Ala-CH2-F, a PFMK cathepsin enzyme inhibitor.
- 62Schlichting, I.; Berendzen, J.; Chu, K.; Stock, A. M.; Maves, S. A.; Benson, D. E.; Sweet, R. M.; Ringe, D.; Petsko, G. A.; Sligar, S. G. The catalytic pathway of cytochrome p450cam at atomic resolution. Science 2000, 287, 1615– 1622, DOI: 10.1126/science.287.5458.1615[Crossref], [PubMed], [CAS], Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXhs1Ortbw%253D&md5=468f33a36d3faf0f8ae2476b3e3a640bThe catalytic pathway of cytochrome P450cam at atomic resolutionSchlichting, Ilme; Berendzen, Joel; Chu, Kelvin; Stock, Ann M.; Maves, Shelley A.; Benson, David E.; Sweet, Robert M.; Ringe, Dagmar; Petsko, Gregory A.; Sligar, Stephen G.Science (Washington, D. C.) (2000), 287 (5458), 1615-1622CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Members of the cytochrome P 450 superfamily catalyze the addn. of mol. oxygen to nonactivated hydrocarbons at physiol. temp., a reaction that requires high temp. to proceed to the absence of a catalyst. Structures were obtained for three intermediates in the hydroxylation reaction of camphor by P450cam with trapping techniques and cryo-crystallog. The structure of the ferrous dioxygen adduct of P450cam was detd. with 0.91 angstrom wavelength x-rays; irradn. with 1.5 angstrom x-rays results in breakdown of the dioxygen mol. to an intermediate that would be consistent with an oxyferryl species. The structures show conformational changes in several important residues and reveal a network of bound water mols. that may provide the protons needed for the reaction.
- 63Qiu, Z.; Kuhn, B.; Aebi, J.; Lin, X.; Ding, H.; Zhou, Z.; Xu, Z.; Xu, D.; Han, L.; Liu, C.; Qiu, H.; Zhang, Y.; Haap, W.; Riemer, C.; Stahl, M.; Qin, N.; Shen, H. C.; Tang, G. Discovery of fluoromethylketone-based peptidomimetics as covalent ATG4B (Autophagin-1) inhibitors. ACS Med. Chem. Lett. 2016, 7, 802– 806, DOI: 10.1021/acsmedchemlett.6b00208[ACS Full Text], [CAS], Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVKisLrL&md5=5068eb96c6fae28342766f54010c5ebeDiscovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) InhibitorsQiu, Zongxing; Kuhn, Bernd; Aebi, Johannes; Lin, Xianfeng; Ding, Haiyuan; Zhou, Zheng; Xu, Zhiheng; Xu, Danqing; Han, Li; Liu, Cheng; Qiu, Hongxia; Zhang, Yuxia; Haap, Wolfgang; Riemer, Claus; Stahl, Martin; Qin, Ning; Shen, Hong C.; Tang, GuozhiACS Medicinal Chemistry Letters (2016), 7 (8), 802-806CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)ATG4B or autophagin-1 is a cysteine protease that cleaves ATG8 family proteins. ATG4B plays essential roles in the autophagosome formation and the autophagy pathway. Herein we disclose the design and structural modifications of a series of fluoromethylketone (FMK)-based peptidomimetics as highly potent ATG4B inhibitors. Their structure-activity relationship (SAR) and protease selectivity are also discussed.
- 64Davies, C. W.; Chaney, J.; Korbel, G.; Ringe, D.; Petsko, G. A.; Ploegh, H.; Das, C. The co-crystal structure of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) with a tripeptide fluoromethyl ketone (Z-VAE(OMe)-FMK). Bioorg. Med. Chem. Lett. 2012, 22, 3900– 3904, DOI: 10.1016/j.bmcl.2012.04.124[Crossref], [PubMed], [CAS], Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XntlSrs7k%253D&md5=1c5dd6939599cd6dc7582871d039209cThe co-crystal structure of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) with a tripeptide fluoromethyl ketone (Z-VAE(OMe)-FMK)Davies, Christopher W.; Chaney, Joseph; Korbel, Gregory; Ringe, Dagmar; Petsko, Gregory A.; Ploegh, Hidde; Das, ChittaranjanBioorganic & Medicinal Chemistry Letters (2012), 22 (12), 3900-3904CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)UCHL1 is a 223 amino acid member of the UCH family of deubiquitinating enzymes (DUBs), found abundantly and exclusively expressed in neurons and the testis in normal tissues. Two naturally occurring variants of UCHL1 are directly involved in Parkinson's disease (PD). Not only has UCHL1 been linked to PD, but it has oncogenic properties, having been found abnormally expressed in lung, pancreatic, and colorectal cancers. Although inhibitors of UCHL1 have been described previously the co-crystal structure of the enzyme bound to any inhibitor has not been reported. Herein, we report the x-ray structure of UCHL1 co-crystd. with a peptide-based fluoromethylketone inhibitor, Z-VAE(OMe)-FMK (VAEFMK) at 2.35 Å resoln. The co-crystal structure reveals that the inhibitor binds in the active-site cleft, irreversibly modifying the active-site cysteine; however, the catalytic histidine is still misaligned as seen in the native structure, suggesting that the inhibitor binds to an inactive form of the enzyme. Our structure also reveals that the inhibitor approaches the active-site cleft from the opposite side of the crossover loop as compared to the direction of approach of ubiquitin's C-terminal tail, thereby occupying the P1' (leaving group) site, a binding site perhaps used by the unknown C-terminal extension of ubiquitin in the actual in vivo substrate(s) of UCHL1. This structure provides a view of mol. contacts at the active-site cleft between the inhibitor and the enzyme as well as furnishing structural information needed to facilitate further design of inhibitors targeted to UCHL1 with high selectivity and potency.
- 65Powers, J. C.; Asgian, J. L.; Ekici, O. D.; James, K. E. Irreversible inhibitors of serine, cysteine, and threonine proteases. Chem. Rev. 2002, 102, 4639– 4750, DOI: 10.1021/cr010182v[ACS Full Text], [CAS], Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XosFSnsr4%253D&md5=eb21ff0297b87537a7d459c92f13c75dIrreversible inhibitors of serine, cysteine, and threonine proteasesPowers, James C.; Asgian, Juliana L.; Ekici, Oezlem Dogan; James, Karen EllisChemical Reviews (Washington, DC, United States) (2002), 102 (12), 4639-4750CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Topics discussed include alkylating, acylating, phosphonylating, and sulfonylating agents.
- 66Heimann, D.; Borgel, F.; de Vries, H.; Bachmann, K.; Rose, V. E.; Frehland, B.; Schepmann, D.; Heitman, L. H.; Wunsch, B. Optimization of pharmacokinetic properties by modification of a carbazole-based cannabinoid receptor subtype 2 (CB2) ligand. Eur. J. Med. Chem. 2018, 143, 1436– 1447, DOI: 10.1016/j.ejmech.2017.10.049[Crossref], [PubMed], [CAS], Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslyrt7jN&md5=4a5e0eb0666d6be4bfd948f2abe58ac9Optimization of pharmacokinetic properties by modification of a carbazole-based cannabinoid receptor subtype 2 (CB2) ligandHeimann, Dominik; Boergel, Frederik; de Vries, Henk; Bachmann, Kim; Rose, Victoria E.; Frehland, Bastian; Schepmann, Dirk; Heitman, Laura H.; Wuensch, BernhardEuropean Journal of Medicinal Chemistry (2018), 143 (), 1436-1447CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Recently, the development of the fluorinated PET tracer [18F]1a for imaging of CB2 receptors in the central nervous system was reported. [18F]1a showed high CB2 affinity and selectivity over the CB1 subtype, but rapid biotransformation in mice. In addn. to the amide hydrolysis, oxidative N-dealkylation and carbazole oxidn. were postulated as main metabolic pathways. Based on these results, novel carbazole derivs. with addnl. 6-substituents (23a, 24a), modified hydrogenation state (26a) and enlarged fluoroalkyl substituent (13a, 13b) were synthesized and pharmacol. evaluated. The key step in the synthesis of substituted carbazoles 23a, 24a and 26a was a Fischer indole synthesis. Nucleophilic substitution of tosylated lactate 5 by carbazole anion provided the fluoroisopropyl derivs. 13a and 13b. Partial hydrogenation of the arom. carbazole system (26a) was not tolerated by the CB2 receptor. A methylsulfonyl moiety in 6-position (24a) led to considerably reduced CB2 affinity, whereas a 6-methoxy moiety (23a) was well tolerated. An addnl. Me moiety in the fluoroethyl side chain of 1a resulted in fluoroisopropyl derivs. 13 with unchanged high CB2 affinity and CB2: CB1 selectivity. Compared with the fluoroethyl deriv. 1a, the carbazole N-atom of the fluoroisopropyl deriv. 13a (Ki(CB2) = 2.9 nM) is better shielded against the attack of CYP enzymes as formation of N-oxides was not obsd. and N-dealkylation took place to a less amt.
- 67Frolova, A. D.; Kuznetsova, E. E. [Transformations of fluorinated ketones in the organism]. Gig. Tr. Prof. Zabol. 1971, 15, 57– 59[PubMed], [CAS], Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3MXks1WmtLY%253D&md5=bc8bc7a38faed36cde566f7d633cb291Transformation of fluorinated ketones in the organismFrolova, A. D.; Kuznetsova, E. E.Gigiena Truda i Professional'nye Zabolevaniya (1971), 15 (6), 57-9CODEN: GTPZAB; ISSN:0016-9919.Acute intoxication by monofluoroacetone in rats caused accumulation of citric acid in the kidneys (fluoroacetate type of action). A toxic concn. of perfluoroacetone dihydrate increased in 6 hr the excretion of glucuronides by urine. The synthesis of fluorinated keto acid or fluorinated secondary alc. is presumed.
- 68Mazurek, U.; Koszinowski, K.; Schwarz, H. C–F bond activation in fluorinated carbonyl compounds by chromium monocations in the gas phase. Organometallics 2003, 22, 218– 225, DOI: 10.1021/om020646v[ACS Full Text], [CAS], Google Scholar68https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xps1Wqtbs%253D&md5=4bb2a82960e596a8697d2a59cdba6c1bC-F Bond Activation in Fluorinated Carbonyl Compounds by Chromium Monocations in the Gas PhaseMazurek, Ulf; Koszinowski, Konrad; Schwarz, HelmutOrganometallics (2003), 22 (2), 218-225CODEN: ORGND7; ISSN:0276-7333. (American Chemical Society)The Cr+-assisted hydrolytic C-F bond activation in the gas phase reported recently for hexafluoroacetone applies also to other fluorinated carbonyl compds. C-F bond hydrolysis is obsd. for monofluoroacetone, 1,1,1-trifluoroacetone, pentafluorobenzaldehyde, and 2,3,4,5,6-pentafluoroacetophenone. However, the diversity of the carbonyl group's substituents is paralleled by an increase of the hitherto small no. of reaction channels, thus allowing for alternative ways of C-F bond activation as well. Both complexation and C-F bond activation of the org. substrates in the gas phase have been investigated by FT-ICR mass spectrometry.
- 69Arellano, M.; Malet-Martino, M.; Martino, R.; Gires, P. The anti-cancer drug 5-fluorouracil is metabolized by the isolated perfused rat liver and in rats into highly toxic fluoroacetate. Br. J. Cancer 1998, 77, 79– 86, DOI: 10.1038/bjc.1998.12[Crossref], [PubMed], [CAS], Google Scholar69https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXoslertg%253D%253D&md5=64430ef44e3f0016a1617e093fa36c14The anti-cancer drug 5-fluorouracil is metabolized by the isolated perfused rat liver and in rats into highly toxic fluoroacetateArellano, M.; Malet-Martino, M.; Martino, R.; Gires, P.British Journal of Cancer (1998), 77 (1), 79-86CODEN: BJCAAI; ISSN:0007-0920. (Churchill Livingstone)The authors report the first demonstration of the biotransformation of the anti-cancer drug 5-fluorouracil (FU) into two new metabolites, α-fluoro-β-hydroxypropionic acid (FHPA) and fluoroacetate (FAC), in the isolated perfused rat liver (IPRL) and in the rat in vivo. IPRL was perfused with solns. of pure FU at two doses, 15 or 45 mg kg-1 body wt., and rats were injected i.p. with 180 mg of FU kg-1 body wt. Fluorine-19 NMR anal. of perfusates from IPRL and rat urine showed the presence of the normal metabolites of FU and low amts. of FHPA (0.4% or 0.1% of injected FU in perfusates from IPRL treated with 15 or 45 mg of FU kg-1 body wt., resp.; 0.08% of the injected FU in rat urine) and FAC (0.1% or 0.03% of injected FU in perfusates from IPRL treated with 15 or 45 mg of FU kg-1 body wt., resp.; 0.003% of the injected FU in rat urine). IPRL was also perfused with a soln. of α-fluoro-β-alanine (FBAL) hydrochloride at 16.6 mg kg-1 body wt. dose equiv. to 15 mg of FU kg-1 body wt. Low amts. of FHPA (0.2% of injected FBAL) and FAC (0.07%) were detected in perfusates, thus demonstrating that FHPA and FAC arise from FBAL catabolism. As FAC is a well-known cardiotoxic poison, and FHPA is also cardiotoxic at high doses, the cardiotoxicity of FU might stem from at least two sources. The first one, established in previous papers, is the presence in com. solns. of FU of degrdn. products of FU that are metabolized into FHPA and FAC; these are formed over time in the basic medium necessary to dissolve the drug. The second, demonstrated in the present study, is the metab. of FU itself into the same compds.
- 70Malet-Martino, M.; Gilard, V.; Desmoulin, F.; Martino, R. Fluorine nuclear magnetic resonance spectroscopy of human biofluids in the field of metabolic studies of anticancer and antifungal fluoropyrimidine drugs. Clin. Chim. Acta 2006, 366, 61– 73, DOI: 10.1016/j.cca.2005.10.013[Crossref], [PubMed], [CAS], Google Scholar70https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XitVKhtb0%253D&md5=d064f058075c536e08c00faf85dd4ac2Fluorine nuclear magnetic resonance spectroscopy of human biofluids in the field of metabolic studies of anticancer and antifungal fluoropyrimidine drugsMalet-Martino, Myriam; Gilard, Veronique; Desmoulin, Franck; Martino, RobertClinica Chimica Acta (2006), 366 (1-2), 61-73CODEN: CCATAR; ISSN:0009-8981. (Elsevier Ltd.)A review with refs. Fluorine-19 NMR (19F NMR) spectroscopy provides a highly specific tool for the detection, identification and quantification of fluorine-contg. drugs and their metabolites in biofluids. The value and difficulties encountered in investigations on drug metab. are first discussed. Then the metab. of three fluoropyrimidines in clin. use, 5-fluorouracil, 5-fluorocytosine and capecitabine are reported. Besides the parent drug and the already known fluorinated metabolites, 12 new metabolites were identified for the first time with 19F NMR in human biofluids. Nine of them can only be obsd. with this technique: fluoride ion, N-carboxy-α-fluoro-β-alanine, α-fluoro-β-alanine conjugate with deoxycholic acid, 2-fluoro-3-hydroxypropanoic acid, fluoroacetic acid, O2-β-glucuronide of fluorocytosine, fluoroacetaldehyde hydrate and its adduct with urea, fluoromalonic acid semi-aldehyde adducts with urea. This emphasizes the high anal. potential of 19F NMR for the furtherance in the understanding of fluoropyrimidine catabolic pathways. 19F NMR should also play a role in the therapeutic monitoring of FU and its prodrugs in specific groups of patients, e.g. hemodialyzed patients or patients with deficiency in FU catabolic enzymes.
- 71Derissen, E. J.; Jacobs, B. A.; Huitema, A. D.; Rosing, H.; Schellens, J. H.; Beijnen, J. H. Exploring the intracellular pharmacokinetics of the 5-fluorouracil nucleotides during capecitabine treatment. Br. J. Clin. Pharmacol. 2016, 81, 949– 957, DOI: 10.1111/bcp.12877[Crossref], [PubMed], [CAS], Google Scholar71Exploring the intracellular pharmacokinetics of the 5-fluorouracil nucleotides during capecitabine treatmentDerissen, Ellen J. B.; Jacobs, Bart A. W.; Huitema, Alwin D. R.; Rosing, Hilde; Schellens, Jan H. M.; Beijnen, Jos H.British Journal of Clinical Pharmacology (2016), 81 (5), 949-957CODEN: BCPHBM; ISSN:1365-2125. (Wiley-Blackwell)Aim : Three intracellularly formed metabolites are responsible for the antineoplastic effect of capecitabine: 5-fluorouridine 5'-triphosphate (FUTP), 5-fluoro-2'-deoxyuridine 5'-triphosphate (FdUTP), and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). The objective of this study was to explore the pharmacokinetics of these intracellular metabolites during capecitabine treatment. Methods : Serial plasma and peripheral blood mononuclear cell (PBMC) samples were collected from 13 patients treated with capecitabine 1000 mg QD (group A) and eight patients receiving capecitabine 850 mg m-2 BID for fourteen days, every three weeks (group B). Samples were collected on day 1 and, for four patients of group B, also on day 14. The capecitabine and 5-fluorouracil (5-FU) plasma concns. and intracellular metabolite concns. were detd. using LC-MS/MS. Pharmacokinetic parameters were estd. using non-compartmental anal. Results : Only FUTP could be measured in the PBMC samples. The FdUTP and FdUMP concns. were below the detection limits (LOD). No significant correlation was found between the plasma 5-FU and intracellular FUTP exposure. The FUTP concn.-time profiles demonstrated considerable inter-individual variation and accumulation of the metabolite in PBMCs. FUTP levels ranged betweenhttps://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmtF2mu7s%253D&md5=d31ab3b2043d45bbe86305e934247ab072Desmoulin, F.; Gilard, V.; Malet-Martino, M.; Martino, R. Metabolism of capecitabine, an oral fluorouracil prodrug: (19)F NMR studies in animal models and human urine. Drug Metab. Dispos. 2002, 30, 1221– 1229, DOI: 10.1124/dmd.30.11.1221[Crossref], [PubMed], [CAS], Google Scholar72https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XotFyku74%253D&md5=ca4db01fd109f974459848cd82a6e974Metabolism of capecitabine, an oral fluorouracil prodrug: 19F NMR studies in animal models and human urineDesmoulin, Franck; Gilard, Veronique; Malet-Martino, Myriam; Martino, RobertDrug Metabolism and Disposition (2002), 30 (11), 1221-1229CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)Capecitabine (Xeloda; CAP) is a recently developed oral antineoplastic prodrug of 5-fluorouracil (5-FU) with enhanced tumor selectivity. Previous studies have shown that CAP activation follows a pathway with three enzymic steps and two intermediary metabolites, 5'-deoxy-5-fluorocytidine (5'-DFCR) and 5'-deoxy-5-fluorouridine (5'-DFUR), to form 5-FU preferentially in tumor tissues. In the present work, we investigated all fluorinated compds. present in liver, bile, and perfusate medium of isolated perfused rat liver (IPRL) and in liver, plasma, kidneys, bile, and urine of healthy rats. Moreover, data obtained from rat urine were compared with those from mice and human urine. According to a low cytidine deaminase (3.5.4.5) activity in rats, 5'-DFCR was by far the main product in perfusate medium from IPRL and plasma and urine from rats. Liver and circulating 5'-DFCR in perfusate and plasma equilibrated at the same concn. value in the range 25 to 400 μM, which supports the involvement of es-type nucleoside transporter in the liver. 5'-DFUR and α-fluoro-β-ureidopropionic acid (FUPA) + α-fluoro-β-alanine (FBAL) were the main products in urine of mice, making up 23 to 30% of the administered dose vs. 3 to 4% in rat. In human urine, FUPA + FBAL represented 50% of the administered dose, 5'-DFCR 10%, and 5'-DFUR 7%. Since fluorine-19 NMR spectroscopy gives an overview of all the fluorinated compds. present in a sample, we obsd. the following unreported metabolites of CAP:. 1) 5-Fluorocytosine and its hydroxylated metabolite, 5-fluoro-6-hydroxycytosine,. 2) Fluoride ion,. 3) 2-Fluoro-3-hydroxypropionic acid and fluoroacetate, and. 4) A glucuroconjugate of 5'-DFCR.73Carreras, C. W.; Santi, D. V. The catalytic mechanism and structure of thymidylate synthase. Annu. Rev. Biochem. 1995, 64, 721– 762, DOI: 10.1146/annurev.bi.64.070195.003445[Crossref], [PubMed], [CAS], Google Scholar73https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXmsl2qtrg%253D&md5=3fa2b583e048196a12193b27f9533770The catalytic mechanism and structure of thymidylate synthaseCarreras, Christopher W.; Santi, Daniel V.Annual Review of Biochemistry (1995), 64 (), 721-62CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)A review, with 193 refs. Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the reductive methylation of dUMP by CH2H4folate to produce dTMP and H2folate. Knowledge of the catalytic mechanism and structure of TS has increased substantially over recent years. Major advances were derived from crystal structures of TS bound to various ligands, the ability to overexpress TS in heterologous hosts, and the numerous mutants that have been prepd. and analyzed. These advances, coupled with previous knowledge, have culminated in an in-depth understanding of many important mol. details of the reaction. The authors review aspects of TS catalysis that are most pertinent to understanding the current status of the structure and catalytic mechanism of the enzyme. Included is a discussion of available sources and assays for TS, a description of the enzyme's chem. mechanism and crystal structure, and a summary of data obtained from mutagenesis expts.74Zhang, X.; Zhang, N.; Chen, G.; Turpoff, A.; Ren, H.; Takasugi, J.; Morrill, C.; Zhu, J.; Li, C.; Lennox, W.; Paget, S.; Liu, Y.; Almstead, N.; George Njoroge, F.; Gu, Z.; Komatsu, T.; Clausen, V.; Espiritu, C.; Graci, J.; Colacino, J.; Lahser, F.; Risher, N.; Weetall, M.; Nomeir, A.; Karp, G. M. Discovery of novel HCV inhibitors: synthesis and biological activity of 6-(indol-2-yl)pyridine-3-sulfonamides targeting hepatitis C virus NS4B. Bioorg. Med. Chem. Lett. 2013, 23, 3947– 3953, DOI: 10.1016/j.bmcl.2013.04.049[Crossref], [PubMed], [CAS], Google Scholar74https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXnvFSisrw%253D&md5=c4ea419ba26dea48358a3036caf48abfDiscovery of novel HCV inhibitors: Synthesis and biological activity of 6-(indol-2-yl)pyridine-3-sulfonamides targeting hepatitis C virus NS4BZhang, Xiaoyan; Zhang, Nanjing; Chen, Guangming; Turpoff, Anthony; Ren, Hongyu; Takasugi, James; Morrill, Christie; Zhu, Jin; Li, Chunshi; Lennox, William; Paget, Steven; Liu, Yalei; Almstead, Neil; George Njoroge, F.; Gu, Zhengxian; Komatsu, Takashi; Clausen, Valerie; Espiritu, Christine; Graci, Jason; Colacino, Joseph; Lahser, Fred; Risher, Nicole; Weetall, Marla; Nomeir, Amin; Karp, Gary M.Bioorganic & Medicinal Chemistry Letters (2013), 23 (13), 3947-3953CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)A novel series of 6-(indol-2-yl)pyridine-3-sulfonamides I [R1 = CHF2O, c-Pr; R2 = i-Pr, H, CH(CH2F)2, etc.] was prepd. and evaluated for their ability to inhibit HCV RNA replication in the HCV replicon cell culture assay. Preliminary optimization of this series furnished compds. with low nanomolar potency against the HCV genotype 1b replicon. Among these, compd. I [R1 = CHF2O; R2 = CH(CH2F)2] was identified as a potent HCV replicon inhibitor (EC50 = 4 nM) with a selectivity index with respect to cellular GAPDH of more than 2500. Further, compd. I [R1 = CHF2O; R2 = CH(CH2F)2] had a good pharmacokinetic profile in rats with an IV half-life of 6 h and oral bioavailability (F) of 62%. Selection of HCV replicon resistance identified an amino acid substitution in HCV NS4B that confers resistance to these compds. These compds. hold promise as a new chemotype with anti-HCV activity mediated through an underexploited viral target.75Zhang, N.; Zhang, X.; Zhu, J.; Turpoff, A.; Chen, G.; Morrill, C.; Huang, S.; Lennox, W.; Kakarla, R.; Liu, R.; Li, C.; Ren, H.; Almstead, N.; Venkatraman, S.; Njoroge, F. G.; Gu, Z.; Clausen, V.; Graci, J.; Jung, S. P.; Zheng, Y.; Colacino, J. M.; Lahser, F.; Sheedy, J.; Mollin, A.; Weetall, M.; Nomeir, A.; Karp, G. M. Structure-activity relationship (SAR) optimization of 6-(indol-2-yl)pyridine-3-sulfonamides: identification of potent, selective, and orally bioavailable small molecules targeting hepatitis C (HCV) NS4B. J. Med. Chem. 2014, 57, 2121– 2135, DOI: 10.1021/jm401621g[ACS Full Text], [CAS], Google Scholar75https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVajtbjL&md5=135ebcfa314f96d786711155128b78c4Structure-Activity Relationship (SAR) Optimization of 6-(Indol-2-yl)pyridine-3-sulfonamides: Identification of Potent, Selective, and Orally Bioavailable Small Molecules Targeting Hepatitis C (HCV) NS4BZhang, Nanjing; Zhang, Xiaoyan; Zhu, Jin; Turpoff, Anthony; Chen, Guangming; Morrill, Christie; Huang, Song; Lennox, William; Kakarla, Ramesh; Liu, Ronggang; Li, Chunshi; Ren, Hongyu; Almstead, Neil; Venkatraman, Srikanth; Njoroge, F. George; Gu, Zhengxian; Clausen, Valerie; Graci, Jason; Jung, Stephen P.; Zheng, Yingcong; Colacino, Joseph M.; Lahser, Fred; Sheedy, Josephine; Mollin, Anna; Weetall, Marla; Nomeir, Amin; Karp, Gary M.Journal of Medicinal Chemistry (2014), 57 (5), 2121-2135CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Nonracemic (aminosulfonyl)pyridinyl indolecarbonitriles such as I were prepd. as inhibitors of the hepatitis C viral protein NS4B for use as antihepatitis C agents. The substitution patterns on the indole rings were modified to limit oxidative metab. of the indolecarbonitriles and to avoid potential liver damage and cytochrome P450 inhibition; the compds. were also optimized to improve their oral bioavailabilities. I was potent against the HCV 1b replicon, with an EC50 value of 2 nM and a selectivity of >5000 with respect to cellular glyceraldehyde-3-phosphate dehydrogenase. I had a favorable pharmacokinetic profile with oral bioavailabilities of 62%, 78%, and 18% in rats, dogs, and monkeys, resp., and favorable tissue distribution properties (liver to plasma exposure ratio in rats of 25).76Appelman, L. M.; Woutersen, R. A.; Feron, V. J. Inhalation toxicity of acetaldehyde in rats. I. Acute and subacute studies. Toxicology 1982, 23, 293– 307, DOI: 10.1016/0300-483X(82)90068-3[Crossref], [PubMed], [CAS], Google Scholar76https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XkvVKmu74%253D&md5=8af732af35478d6f36313c8342c7bc15Inhalation toxicity of acetaldehyde in rats. I. Acute and subacute studiesAppelman, L. M.; Woutersen, R. A.; Feron, V. J.Toxicology (1982), 23 (4), 293-307CODEN: TXCYAC; ISSN:0300-483X.The 4-h median lethal concn. of AcH [75-07-0] in rats was 13,300 ppm (24.0 g/m3 air). In a 4-wk study, groups of male and female rats were exposed to 0, 400, 1000, 2200 or 5000 ppm AcH for 6 h/day, 5 days/wk. Treatment-related changes obsd. at the 5000 ppm level included dyspnea and excitation during the 1st 30 min of each exposure, yellow-brown fur, severe growth retardation, more neutrophils and less lymphocytes in the blood, a reduced prodn. of urine with a high d., increased lung wts., and severe degenerative, hyperplastic and metaplastic changes of the nasal, laryngeal and tracheal epithelium. Major lesions seen at 1000 and 2200 ppm comprised growth retardation and an increased prodn. of urine in males, slight to moderate degeneration with or without hyper- and metaplasia of the nasal epithelium, and only at 220 ppm, minimal epithelial changes in the larynx and trachea. The only change obsd. at the 400 ppm level that could be attributed to AcH was slight degeneration of the nasal olfactory epithelium seen as loss of microvilli and thinning and disarrangement of the layer of epithelial cells.77Woutersen, R. A.; Appelman, L. M.; Van der Heijden, C. A. Inhalation toxicity of acetaldehyde in rats. II. Carcinogenicity study: interim results after 15 months. Toxicology 1984, 31, 123– 133, DOI: 10.1016/0300-483X(84)90004-0[Crossref], [PubMed], [CAS], Google Scholar77https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXltVGmsbY%253D&md5=8a65ee868ccab29ae05d811a3898a1a1Inhalation toxicity of acetaldehyde in rats. II. Carcinogenicity study: interim results after 15 monthsWoutersen, R. A.; Appelman, L. M.; Feron, V. J.; Van der Heijden, C. A.Toxicology (1984), 31 (2), 123-33CODEN: TXCYAC; ISSN:0300-483X.Male and female rats were exposed to AcH [75-07-0] vapor at 0, 750, 1500 and 3000/1000 ppm during 6 h/day, 5 days/wk for up to 27 mo. During the 1st 15 mo of the study, major compd.-related lesions occurred in the nose and larynx. The nasal lesions comprised: (1) degenerative changes of the olfactory epithelium at all dose levels, frequently accompanied by focal hyperplasia of basal cells and thickening of the submucosa with loss of Bowman's glands and nerve bundles in the dorsomedial region, (2) stratified squamous metaplasia of the respiratory epithelium lining the caudoventral part of the nasal septum and the inner aspect of the ventral endoturbinates often accompanied by severe keratinization and occasionally by papillomatous hyperplasia, almost exclusively obsd. at the top-dose level, and (3) malignant tumors (squamous cell carcinomas and adenocarcinomas) at all dose levels. Hyperplasia and keratinized stratified squamous metaplasia of the laryngeal epithelium were seen at the 2 highest dose levels.78Woutersen, R. A.; Appelman, L. M.; Van Garderen-Hoetmer, A.; Feron, V. J. Inhalation toxicity of acetaldehyde in rats. III. Carcinogenicity study. Toxicology 1986, 41, 213– 231, DOI: 10.1016/0300-483X(86)90201-5[Crossref], [PubMed], [CAS], Google Scholar78https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28Xmt1Skt70%253D&md5=6880976e51089259ca4dfc12bd17ea9bInhalation toxicity of acetaldehyde in rats. III. Carcinogenicity studyWoutersen, R. A.; Appelman, L. M.; Van Garderen-Hoetmer, A.; Feron, V. J.Toxicology (1986), 41 (2), 213-31CODEN: TXCYAC; ISSN:0300-483X.Male and female rats were exposed to AcH [75-07-0] vapor at nominal concns. of 0, 750, 1500, or 3000 ppm (gradually reduced to 1000 ppm during the 1st 52 wk) during 6 h/day, 5 days/wk for ≤28 mo. Major compd.-related effects included increased mortality, growth retardation, nasal tumors, and nonneoplastic nasal changes in each of the test groups. The treatment-related nasal changes comprised: (1) degeneration, hyperplasia, metaplasia, and adenocarcinomas of the olfactory epithelium at all exposure levels; (2) squamous metaplasia accompanied by slight to severe keratinization and squamous cell carcinomas of the respiratory epithelium at the 2 highest exposure levels; and (3) slight to severe rhinitis and sinusitis in top-concn. rats. In the larynx hyperplasia and keratinized squamous metaplasia of the epithelium in the vocal cord region were seen in many rats of the mid- and top-concn. groups. One female rat of the 1500-ppm group had developed a laryngeal carcinoma in situ. Evidently, AcH is both cytotoxic and carcinogenic to the nasal mucosa of rats.79Setshedi, M.; Wands, J. R.; de la Monte, S. M. Acetaldehyde adducts in alcoholic liver disease. Oxid. Med. Cell. Longevity 2010, 3, 178– 185, DOI: 10.4161/oxim.3.3.12288[Crossref], [PubMed], [CAS], Google Scholar79https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3cjlsF2ntg%253D%253D&md5=52da45f51f23fc86bfa7f854fdb70263Acetaldehyde adducts in alcoholic liver diseaseSetshedi Mashiko; Wands Jack R; Monte Suzanne M de laOxidative medicine and cellular longevity (2010), 3 (3), 178-85 ISSN:.Chronic alcohol abuse causes liver disease that progresses from simple steatosis through stages of steatohepatitis, fibrosis, cirrhosis, and eventually hepatic failure. In addition, chronic alcoholic liver disease (ALD), with or without cirrhosis, increases risk for hepatocellular carcinoma (HCC). Acetaldehyde, a major toxic metabolite, is one of the principal culprits mediating fibrogenic and mutagenic effects of alcohol in the liver. Mechanistically, acetaldehyde promotes adduct formation, leading to functional impairments of key proteins, including enzymes, as well as DNA damage, which promotes mutagenesis. Why certain individuals who heavily abuse alcohol, develop HCC (7.2-15%) versus cirrhosis (15-20%) is not known, but genetics and co-existing viral infection are considered pathogenic factors. Moreover, adverse effects of acetaldehyde on the cardiovascular system and hematologic systems leading to ischemia, heart failure, and coagulation disorders, can exacerbate hepatic injury and increase risk for liver failure. Herein, we review the role of acetaldehyde adducts in the pathogenesis of chronic ALD and HCC.80Nagasawa, H. T.; Valentekovich, R. J.; Nagasawa, S. G.; Nagasawa, R. H. Sequestration and elimination of toxic aldehydes. Chem. Res. Toxicol. 2020, DOI: 10.1021/acs.chemrestox.9b0037381Ernstgard, L.; Iregren, A.; Sjogren, B.; Johanson, G. Acute effects of exposure to vapours of acetic acid in humans. Toxicol. Lett. 2006, 165, 22– 30, DOI: 10.1016/j.toxlet.2006.01.010[Crossref], [PubMed], [CAS], Google Scholar81https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XlvF2gu74%253D&md5=04ea02fe480a664b494cc0ff8525619aAcute effects of exposure to vapours of acetic acid in humansErnstgard, Lena; Iregren, Anders; Sjoegren, Bengt; Johanson, GunnarToxicology Letters (2006), 165 (1), 22-30CODEN: TOLED5; ISSN:0378-4274. (Elsevier B.V.)Acetic acid is used in plastics, chem. and pharmaceutical industries. Despite a widespread use, information of possible health effects is sparse. The aim of this study was to evaluate acute irritation during controlled exposure to vapors of acetic acid. Six female and 6 male healthy volunteers were exposed to 0 ppm (control exposure), 5 and 10 ppm acetic acid vapor for 2 h at rest in a balanced order. Subjective ratings of nasal irritation and smell increased significantly with exposure level. Except for smell, all av. ratings at 10 ppm were at the lower end of the 0-100 mm visual analog scale, and did not exceed the verbal expression "somewhat" (26 mm). No effects on pulmonary function, nasal swelling, nasal airway resistance or plasma inflammatory markers (C-reactive protein, and interleukin-6), measured before and after exposure, were seen. There was a non-significant tendency to increased blinking frequency, as measured continuously during exposure, after exposure to 10 ppm acetic acid. In conclusion, our study suggests a mild irritative effect at 10 ppm acetic acid.82Zeiger, E.; Anderson, B.; Haworth, S.; Lawlor, T.; Mortelmans, K. Salmonella mutagenicity tests: V. Results from the testing of 311 chemicals. Environ. Mol. Mutagen. 1992, 19 (S21), 2– 141, DOI: 10.1002/em.2850190603[Crossref], [PubMed], [CAS], Google Scholar82https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xks1Cmurw%253D&md5=ef3f19f34f891ad579448473326a7182Salmonella mutagenicity tests: V. Results from the testing of 311 chemicalsZeiger, Errol; Anderson, Beth; Haworth, Steve; Lawlor, Timothy; Mortelmans, KristienEnvironmental and Molecular Mutagenesis (1992), 19 (Suppl. 21), 2-141CODEN: EMMUEG; ISSN:0893-6692.Three hundred eleven chems. were tested under code, for mutagenicity, in S. typhimurium; 35 of the chems. were tested more than once in the same or different labs. The tests were conducted using a preincubation protocol in the absence of exogenous metabolic activation, and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters. Some of the volatile chems. were also tested in desiccators. A total of 120 chems. were mutagenic or weakly mutagenic, 3 were judged questionable, and 172 were nonmutagenic. The remaining 16 chems. produced different responses in the two or three labs. in which they were tested. The results and data from these tests are presented.83Morita, T.; Takeda, K.; Okumura, K. Evaluation of clastogenicity of formic acid, acetic acid and lactic acid on cultured mammalian cells. Mutat. Res., Genet. Toxicol. Test. 1990, 240, 195– 202, DOI: 10.1016/0165-1218(90)90058-A[Crossref], [PubMed], [CAS], Google Scholar83https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXhsFemsrs%253D&md5=5c1f988071dede0a3d0f84b6e3272126Evaluation of clastogenicity of formic acid, acetic acid, and lactic acid on cultured mammalian cellsMorita, Takeshi; Takeda, Kenzo; Okumura, KazuoMutation Research, Genetic Toxicology Testing (1990), 240 (3), 195-202CODEN: MRGTE4; ISSN:0165-1218.Using Chinese hamster ovary K1 cells, chromosomal aberration tests were carried out with formic acid, acetic acid and lactic acid, and the relation between the pH of the medium and the clastogenic activity was examd. The medium used was Ham's F12 supplemented with 17 mM NaHCO3 and 10% fetal calf serum. All the acids induced chromosomal aberrations at the initial pH of ca. 6.0 or below (∼10-14 mM of each acid) both with and without S9 mix. Exposure of cells to about pH 5.7 or below (∼12-16 mM of each acid) was toxic. When the culture medium was first acidified with each of the acids and then neutralized to pH 6.4 or pH 7.2 with NaOH, no clastogenic activity was obsd. Using F12 medium supplemented with 34 mM NaHCO3 as a buffer, no clastogenic activity was obsd. at doses up to 25 mM of the acids (initial pH 5.8-6.0). However, ∼10% of the cells had aberrations at pH 5.7 or below (27.5-32.5 mM of each acid). Furthermore, when 30 mM HEPES was used as a buffer, chromosomal aberrations were not induced at doses up to 20 mM formic acid and acetic acid (initial pH 7.0-7.1), and at doses up to 30 mM lactic acid (initial pH 6.6). In the initial pH range of 6.4-6.7 (25-32.5 mM of each acid), chromosomal aberrations were obsd. The above results show that the acids themselves are nonclastogenic, and the pseudopos. reactions attributable to nonphysiol. pH could be eliminated by either neutralization of the medium or enhancement of its buffering ability.84Rotstein, J. B.; Slaga, T. J. Acetic acid, a potent agent of tumor progression in the multistage mouse skin model for chemical carcinogenesis. Cancer Lett. 1988, 42, 87– 90, DOI: 10.1016/0304-3835(88)90243-1[Crossref], [PubMed], [CAS], Google Scholar84https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXjsFWgtQ%253D%253D&md5=833c940fb86167f0d7e62f696656364aAcetic acid, a potent agent of tumor progression in the multistage mouse skin model for chemical carcinogenesisRotstein, J. B.; Slaga, T. J.Cancer Letters (Shannon, Ireland) (1988), 42 (1-2), 87-90CODEN: CALEDQ; ISSN:0304-3835.Acetic acid, a very weak tumor promoter in the multistage mouse skin model for chem. carcinogenesis, was found to be very effective at enhancing cancer development, when applied during the progression phase of the model. Papilloma-bearing mice when repeatedly treated with acetic acid had a greater carcinoma incidence and a greater conversion of papillomas to carcinomas than vehicle-treated mice. Selective cytotoxicity is discussed as a possible mechanism.85Hunter, E. S., 3rd; Rogers, E. H.; Schmid, J. E.; Richard, A. Comparative effects of haloacetic acids in whole embryo culture. Teratology 1996, 54, 57– 64, DOI: 10.1002/(SICI)1096-9926(199606)54:2<57::AID-TERA1>3.0.CO;2-1[Crossref], [PubMed], [CAS], Google Scholar85https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xntleju7w%253D&md5=8ee90401496b0b2652177e2858449941Comparative effects of haloacetic acids in whole embryo cultureHunter, E, Sidney, III; Rogers, E. H.; Schmid, J. E.; Richard, A.Teratology (1996), 54 (2), 57-64CODEN: TJADAB; ISSN:0040-3709. (Wiley-Liss)Three-6 somite staged CD-1 mouse embryos were exposed to acetic acid (AA) or mono- (M), di- (D), and tri- (T) substituted fluoro-(F), chloro- (C), or bromo- (B) acetic acids in whole embryo culture to evaluate the effects of these agents on development. A 24 h exposure to the haloacetic acids produced dysmorphogenesis. Effects on neural tube development ranged from prosencephalic hypoplasia to non-closure defects throughout the cranial region. Exposure to the haloacetic acid affected optic development, produced malpositioned and/or hypoplastic pharyngeal arches, and resulted in perturbation of heart development. To det. the relative toxicities of these agents, benchmark concns. were calcd. as the lower 95% confidence interval of the concn. that produced a 5% increase in neural tube defects. The benchmark concns. occurred over a wide range with DFA (5912.6 μM) and MBA (2.7 μM) at the extremes. Using the benchmark concns. to compare the chems. gives a ranking of the agents in order of increasing potency as: DFA < TFA < DCA < AA < TBA ≤ TCA < DBA < MCA < MBA. TCA and DCA have demonstrated ability to disrupt development in vivo but were among the least potent haloacetic acids in vitro. Because of the potential for widespread exposure to haloacetic acids in drinking water and the incomplete toxicity profile of these chems., further work on their developmental effects is warranted.86Richard, A. M.; Hunter, E. S., 3rd. Quantitative structure-activity relationships for the developmental toxicity of haloacetic acids in mammalian whole embryo culture. Teratology 1996, 53, 352– 360, DOI: 10.1002/(SICI)1096-9926(199606)53:6<352::AID-TERA6>3.0.CO;2-1[Crossref], [PubMed], [CAS], Google Scholar86https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xms1Gqtrk%253D&md5=29441116eaf30d4985e8a5c7084021e6Quantitative structure-activity relationships for the developmental toxicity of haloacetic acids in mammalian whole embryo cultureRichard, Ann M.; Hunter, E. Sidney, IIITeratology (1996), 53 (6), 352-360CODEN: TJADAB; ISSN:0040-3709. (Wiley-Liss)Developmental toxicity in mouse whole embryo culture assay has been reported for acetic acid (AA) and a series of ten haloacetic acids, including mono-, di-, tri-fluoro (MFA, DFA, TFA), chloro (MCA, DCA, TCA), bromo (MBA, DBA, TBA), and monoiodo (MIA) acetic acids. Benchmark concns. (BCm), calcd. as the lower 95% confidence limit of molar acid concn. producing a 5% increase in embryos with neural tube defects, provided potency ests. for development of quant. structure-activity relationships (QSARs). The best overall regression was obtained for the ten halo-acids (excluding AA) and related log(1/BCm) to the energy of the LUMO (Elumo) and acid dissocn. const. (pKa) with a correlation coeff. of r = 0.97, and a sample size-adjusted r2 = 0.92. This QSAR suggested a common basis for the mechanism of HA activity, which would imply additivity for mixts. of these acids. Examn. of QSARs for subsets of the total data set (e.g., monohaloacids) highlighted parameter relationships embedded in the total QSAR, helping to unravel the sep. contributions of Elumo and pKa to the overall potency. The relevance of these parameters is discussed in terms of postulated mechanisms of developmental toxicity involving changes in intercellular pH and redox metab. The whole embryo assay results pertain to direct embryo exposure and toxicity without the confounding influence of maternal factors. The resulting QSAR model offers possible insight into the mechanism of embryo toxicity that will hopefully contribute to understanding of the more complex, in vivo teratogenicity problem.87Lloyd, S. C.; Blackburn, D. M.; Foster, P. M. Trifluoroethanol and its oxidative metabolites: comparison of in vivo and in vitro effects in rat testis. Toxicol. Appl. Pharmacol. 1988, 92, 390– 401, DOI: 10.1016/0041-008X(88)90179-2[Crossref], [PubMed], [CAS], Google Scholar87https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXhs1aqs7k%253D&md5=a4b5f844782b0d26d54c7b75f2a88ddaTrifluoroethanol and its oxidative metabolites: comparison of in vivo and in vitro effects in rat testisLloyd, S. C.; Blackburn, D. M.; Foster, P. M. D.Toxicology and Applied Pharmacology (1988), 92 (3), 390-401CODEN: TXAPA9; ISSN:0041-008X.Trifluoroethanol (TFE) and trifluoroacetaldehyde (TFALD) decreased testis wt. 3 days after a single oral dose of 10 mg/kg. In contrast, the administration of trifluoroacetic acid (TFAA) caused no observable testicular effects. Redn. in testis wt. was accompanied by morphol. changes, involving specific damage to pachytene and dividing spermatocytes, and round spermatids. In an in vitro Sertoli/germ cell co-culture system, only TFALD had dose-related effects at concns. of 10-3 and 10-4M. There was increased germ cell loss from the cultures, particularly loss of pachytene and dividing spermatocytes, accompanied by leakage of the pachytene spermatocyte marker enzyme, lactate dehydrogenase-X. TFE and TFAA did not produce these effects in the culture system at concns. equimolar with TFALD. Thus, TFALD may play a crit. role in the development of the testis lesion seen with TFE in vivo. The effects seen both in vivo and in vitro were remarkably similar to those previously reported for another substituted alc. and its metabolites, ethylene glycol monomethyl ether. The 2 series of compds. may have a similar mode of action on rat testis.88Kaminsky, L. S.; Fraser, J. M.; Seaman, M.; Dunbar, D. Rat liver metabolism and toxicity of 2,2,2-trifluoroethanol. Biochem. Pharmacol. 1992, 44, 1829– 1837, DOI: 10.1016/0006-2952(92)90078-W[Crossref], [PubMed], [CAS], Google Scholar88https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXntlCquw%253D%253D&md5=637dd03291a5f959f6f5e10849b81a06Rat liver metabolism and toxicity of 2,2,2-trifluoroethanolKaminsky, Laurence S.; Fraser, Joanne M.; Seaman, Michael; Dunbar, DeborahBiochemical Pharmacology (1992), 44 (9), 1829-37CODEN: BCPCA6; ISSN:0006-2952.2,2,2-Trifluoroethanol (TFE) is a metabolite of anesthetic agents and chlorofluorocarbon alternatives. Its toxicity in rats is a consequence of its metab. to 2,2,2-trifluoroacetaldehyde (TFAld) and then to trifluoroacetic acid (TFAA). The enzymes involved in the toxic metabolic pathway have been investigated in this study. For the reaction of TFE to TFAld, the major hepatic metab. assocd. with toxicity (as assessed by pyrazole-inhibition) was NADPH dependent and occurred in the microsomes, whereas for TFAld conversion to TFAA, NADPH-dependent microsomal metab. was significant, but mitochondrial and cytosolic metab. in the presence of NADPH were also major contributors. NADPH-dependent hepatic microsomal metab. of TFE to TFAld and TFAld to TFAA was inhibited by carbon monoxide, 2-allyl-2-isopropylacetamide, SKF-525A, metyrapone, imidazole, and pyrazole, and both reactions were oxygen dependent. The metab. of TFE to TFAld was inhibited by diethyldithiocarbamate, a specific inhibitor of cytochrome P450E1, and by a monoclonal antibody to P 4502E1, whereas the metab. of TFAld was inhibited by neither agent. Ethanol pretreatment of rats enhanced the Vmax for hepatic microsomal metab. of TFE to TFAld from 5.3 to 9.7 nmol/mg protein/min, while for TFAld to TFAA, the Vmax was increased from 4.3 to 6.5 and the Km was unaffected for both reactions. Phenobarbital pretreatment of the rats did not affect any of these kinetic parameters. Coadministration of ethanol and a LD of TFE very markedly decreased the lethality. Both the lethality (LD50 0.21 to 0.44 g/kg) and the metabolic kinetic parameters [(Vmax/Km)H(Vmax/Km)D = 4.2] were affected markedly when deuterated TFE replaced TFE. In contrast, deuteration of TFAld did not affect its lethality or rates of metab., but did affect its Km. Taken together, these results indicate that P 4502E1 catalyzed toxicity-assocd. hepatic metab. of TFE to TFAld, while TFAld metab. was catalyzed by a P 450 which was not P 4502E1. The hepatic metab. of TFAld was not assocd. with its toxicity, which has been detd. previously to be assocd. with its intestinal metab.89Dowty, M. E.; Hu, G.; Hua, F.; Shilliday, F. B.; Dowty, H. V. Drug design structural alert: formation of trifluoroacetaldehyde through N-dealkylation is linked to testicular lesions in rat. Int. J. Toxicol. 2011, 30, 546– 550, DOI: 10.1177/1091581811413833[Crossref], [PubMed], [CAS], Google Scholar89https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVGktLfM&md5=30f0e43e80ba5d541b48eeee99b2e95bDrug design structural alert: formation of trifluoroacetaldehyde through N-dealkylation is linked to testicular lesions in ratDowty, Martin E.; Hu, George; Hua, Fengmei; Shilliday, F. Barclay; Dowty, Heather V.International Journal of Toxicology (2011), 30 (5), 546-550CODEN: IJTOFN; ISSN:1091-5818. (Sage Publications)In the process of drug design, it is important to consider potential structural alerts that may lead to toxicosis. This work illustrates how using trifluoroethane as a part of a novel chem. entity led to cytochrome P 450 - mediated N-dealkylation and the formation of trifluoroacetaldehyde, a known testicular toxicant, in exploratory safety studies in rats. Testicular toxicosis was noted microscopically in a dose-dependent manner as measured by testicular spermatocytic degeneration and necrosis and excessive intratubular cellular debris in the epididymis. This apparent toxic effect correlated well with the dose-dependent formation of trifluoroacetaldehyde, identified from in vitro rat liver microsome metab. studies. A similar safety study performed with an N-tetrazole substitution in place of the N-trifluoroethane showed no evidence of testicular injury, implicating further the role of trifluoroacetaldehyde in the testicular lesion obsd. These results highlight the relevance of early metabolic and safety testing in assessing potential structural alerts in drug design.90Kanduti, D.; Sterbenk, P.; Artnik, B. Fluoride: A review of use and effects on health. Mater. Sociomed. 2016, 28, 133– 137, DOI: 10.5455/msm.2016.28.133-137[Crossref], [PubMed], [CAS], Google Scholar90https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28bms1amtA%253D%253D&md5=eb0013cea0610f282fb5ff431254e8e1FLUORIDE: A REVIEW OF USE AND EFFECTS ON HEALTHKanduti Domen; Sterbenk Petra; Artnik BarbaraMateria socio-medica (2016), 28 (2), 133-7 ISSN:1512-7680.INTRODUCTION: Appropriate oral health care is fundamental for any individual's health. Dental caries is still one of the major public health problems. The most effective way of caries prevention is the use of fluoride. AIM: The aim of our research was to review the literature about fluoride toxicity and to inform physicians, dentists and public health specialists whether fluoride use is expedient and safe. METHODS: Data we used in our review were systematically searched and collected from web pages and documents published from different international institutions. RESULTS: Fluoride occurs naturally in our environment but we consume it in small amounts. Exposure can occur through dietary intake, respiration and fluoride supplements. The most important factor for fluoride presence in alimentation is fluoridated water. Methods, which led to greater fluoride exposure and lowered caries prevalence, are considered to be one of the greatest accomplishments in the 20th centurys public dental health. During pregnancy, the placenta acts as a barrier. The fluoride, therefore, crosses the placenta in low concentrations. Fluoride can be transmitted through the plasma into the mother's milk; however, the concentration is low. The most important action of fluoride is topical, when it is present in the saliva in the appropriate concentration. The most important effect of fluoride on caries incidence is through its role in the process of remineralization and demineralization of tooth enamel. Acute toxicity can occur after ingesting one or more doses of fluoride over a short time period which then leads to poisoning. Today, poisoning is mainly due to unsupervised ingestion of products for dental and oral hygiene and over-fluoridated water. CONCLUSION: Even though fluoride can be toxic in extremely high concentrations, its topical use is safe. The European Academy of Paediatric Dentistry (EAPD) recommends a preventive topical use of fluoride supplements because of their cariostatic effect.91Ullah, R.; Zafar, M. S.; Shahani, N. Potential fluoride toxicity from oral medicaments: A review. Iran. J. Basic Med. Sci. 2017, 20, 841– 848, DOI: 10.22038/IJBMS.2017.9104[Crossref], [PubMed], [CAS], Google Scholar91https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1M7mtVWjsw%253D%253D&md5=8dc1bca5b45a3fa700cd5ae62745adc9Potential fluoride toxicity from oral medicaments: A reviewUllah Rizwan; Shahani Nazish; Zafar Muhammad Sohail; Zafar Muhammad SohailIranian journal of basic medical sciences (2017), 20 (8), 841-848 ISSN:2008-3866.The beneficial effects of fluoride on human oral health are well studied. There are numerous studies demonstrating that a small amount of fluoride delivered to the oral cavity decreases the prevalence of dental decay and results in stronger teeth and bones. However, ingestion of fluoride more than the recommended limit leads to toxicity and adverse effects. In order to update our understanding of fluoride and its potential toxicity, we have described the mechanisms of fluoride metabolism, toxic effects, and management of fluoride toxicity. The main aim of this review is to highlight the potential adverse effects of fluoride overdose and poorly understood toxicity. In addition, the related clinical significance of fluoride overdose and toxicity has been discussed.92Tu, L. Q.; Wright, P. F.; Rix, C. J.; Ahokas, J. T. Is fluoroacetate-specific defluorinase a glutathione S-transferase?. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 2006, 143, 59– 66, DOI: 10.1016/j.cbpc.2005.12.003[Crossref], [PubMed], [CAS], Google Scholar92https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjtFOku7w%253D&md5=fa0621017a921fd8adb1a73e6a7fe762Is fluoroacetate-specific defluorinase a glutathione S-transferase?Tu, L. Q.; Wright, P. F. A.; Rix, C. J.; Ahokas, J. T.Comparative Biochemistry and Physiology, Part C: Toxicology & Pharmacology (2006), 143C (1), 59-66CODEN: CBPPFK; ISSN:1532-0456. (Elsevier B.V.)Fluoroacetate-specific defluorinase (FSD) is a crit. enzyme in the detoxication of fluoroacetate. This study investigated whether FSD can be classed as a glutathione S-transferase (GST) isoenzyme with a high specificity for fluoroacetate detoxication metab. The majority of FSD and GST activity, using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as GST substrates, in rat liver was cytosolic. GSTT1 specific substrate, EPNP caused a slight non-competitive inhibition of FSD activity. CDNB, a general substrate of GST isoenzyme, was a more potent non-competitive inhibitor of FSD activity. The fluoroacetate defluorination activity by GST isoenzymes was detd. in this study. The results showed that the GSTZ1C had the highest fluoroacetate defluorination activity of the various GST isoenzymes studied, while GSTA2 had a limited activity toward fluoroacetate. The human GSTZ1C recombinant protein then was purified from a human GSTZ1C cDNA clone. Our expts. showed that GSTZ1C catalyzed fluoroacetate defluorination. GSTZ1 shares many of the characteristics of FSD; however, it accounts only for 3% of the total cytosolic FSD activity. GSTZ1C based enzyme kinetic studies has low affinity for fluoroacetate. The evidence suggests that GSTZ1 may not be the major enzyme defluorinating fluoroacetate, but it does detoxify the fluoroacetate. To clarify the identity of enzymes responsible for fluoroacetate detoxication, further studies of the overall FSD activity are needed.93Jitsumori, K.; Omi, R.; Kurihara, T.; Kurata, A.; Mihara, H.; Miyahara, I.; Hirotsu, K.; Esaki, N. X-Ray crystallographic and mutational studies of fluoroacetate dehalogenase from Burkholderia sp. strain FA1. J. Bacteriol. 2009, 191, 2630– 2637, DOI: 10.1128/JB.01654-08[Crossref], [PubMed], [CAS], Google Scholar93https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXltFaku7k%253D&md5=a56bf8ff4136f279044950f5f623f301X-ray crystallographic and mutational studies of fluoroacetate dehalogenase from Burkholderia sp. strain FA1Jitsumori, Keiji; Omi, Rie; Kurihara, Tatsuo; Kurata, Atsushi; Mihara, Hisaaki; Miyahara, Ikuko; Hirotsu, Ken; Esaki, NobuyoshiJournal of Bacteriology (2009), 191 (8), 2630-2637CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)Fluoroacetate dehalogenase (FAc-DEX) catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of a carbon-fluorine bond of an aliph. compd.: the bond energy of the carbon-fluorine bond is among the highest found in natural products. The enzyme also acts on chloroacetate, although much less efficiently. We here detd. the X-ray crystal structure of the enzyme from Burkholderia sp. strain FA1 as the first exptl. detd. three-dimensional structure of fluoroacetate dehalogenase. The enzyme belongs to the α/β hydrolase superfamily and exists as a homodimer. Each subunit consists of core and cap domains. The catalytic triad, Asp104-His271-Asp128, of which Asp104 serves as the catalytic nucleophile, was found in the core domain at the domain interface. The active site was composed of Phe34, Asp104, Arg105, Arg108, Asp128, His271, and Phe272 of the core domain and Tyr147, His149, Trp150, and Tyr212 of the cap domain. An electron d. peak corresponding to a chloride ion was found in the vicinity of the Nε1 atom of Trp150 and the Nε2 atom of His149, suggesting that these are the halide ion acceptors. Site-directed replacement of each of the active-site residues, except for Trp150, by Ala caused the total loss of the activity toward fluoroacetate and chloroacetate, whereas the replacement of Trp150 caused the loss of the activity only toward fluoroacetate. An interaction between Trp150 and the fluorine atom is probably an abs. requirement for the redn. of the activation energy for the cleavage of the carbon-fluorine bond.94Kamachi, T.; Nakayama, T.; Shitamichi, O.; Jitsumori, K.; Kurihara, T.; Esaki, N.; Yoshizawa, K. The catalytic mechanism of fluoroacetate dehalogenase: a computational exploration of biological dehalogenation. Chem. - Eur. J. 2009, 15, 7394– 7403, DOI: 10.1002/chem.200801813[Crossref], [PubMed], [CAS], Google Scholar94https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXovVChsr8%253D&md5=af5089e2d9a528a30ec4a2cdd28eaa2eThe Catalytic Mechanism of Fluoroacetate Dehalogenase: A Computational Exploration of Biological DehalogenationKamachi, Takashi; Nakayama, Tomonori; Shitamichi, Osamu; Jitsumori, Keiji; Kurihara, Tatsuo; Esaki, Nobuyoshi; Yoshizawa, KazunariChemistry - A European Journal (2009), 15 (30), 7394-7403, S7394/1-S7394/56CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)The biol. dehalogenation of fluoroacetate carried out by fluoroacetate dehalogenase is discussed by using quantum mech./mol. mech. (QM/MM) calcns. for a whole-enzyme model of 10 800 atoms. Substrate fluoroacetate is anchored by a hydrogen-bonding network with water mols. and the surrounding amino acid residues of Arg105, Arg108, His149, Trp150, and Tyr212 in the active site in a similar way to haloalkane dehalogenase. Asp104 is likely to act as a nucleophile to attack the α-carbon of fluoroacetate, resulting in the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water mol. to the carbonyl carbon atom. The cleavage of the strong C-F bond is greatly facilitated by the hydrogen-bonding interactions between the leaving fluorine atom and the three amino acid residues of His149, Trp150, and Tyr212. The hydrolysis of the ester intermediate is initiated by a proton transfer from the water mol. to His271 and by the simultaneous nucleophilic attack of the water mol. The transition state and produced tetrahedral intermediate are stabilized by Asp128 and the oxyanion hole composed of Phe34 and Arg105.95Kurihara, T. A mechanistic analysis of enzymatic degradation of organohalogen compounds. Biosci., Biotechnol., Biochem. 2011, 75, 189– 198, DOI: 10.1271/bbb.100746[Crossref], [PubMed], [CAS], Google Scholar95https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjtVWrtro%253D&md5=df0a7c65a24a7c9a098d2db67a38fda3A mechanistic analysis of enzymatic degradation of organohalogen compoundsKurihara, TatsuoBioscience, Biotechnology, and Biochemistry (2011), 75 (2), 189-198CODEN: BBBIEJ; ISSN:0916-8451. (Japan Society for Bioscience, Biotechnology, and Agrochemistry)A review. Enzymes that catalyze the conversion of organohalogen compds. have been attracting a great deal of attention, partly because of their possible applications in environmental technol. and the chem. industry. The authors have studied the mechanisms of enzymic degrdn. of various org. halo acids. In the reaction of L-2-haloacid dehalogenase and fluoroacetate dehalogenase, the carboxylate group of the catalytic Asp residue nucleophilically attacks the α-C atom of the substrates to displace the halogen atom. In the reaction catalyzed by DL-2-haloacid dehalogenase, a water mol. directly attacks the substrate to displace the halogen atom. In the course of studies on the metab. of 2-chloroacrylate, the authors discovered 2 new enzymes. 2-Haloacrylate reductase catalyzes the asym. redn. of 2-haloacrylate to produce L-2-haloalkanoic acid in an NADPH-dependent manner. 2-Haloacrylate hydratase catalyzes the hydration of 2-haloacrylate to produce pyruvate. This enzyme is unique in that it catalyzes the non-redox reaction in an FADH2-dependent manner.96Chan, P. W.; Yakunin, A. F.; Edwards, E. A.; Pai, E. F. Mapping the reaction coordinates of enzymatic defluorination. J. Am. Chem. Soc. 2011, 133, 7461– 7468, DOI: 10.1021/ja200277d[ACS Full Text], [CAS], Google Scholar96https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXltVGgsb8%253D&md5=c25872673b8386d33c29e7a9b3e7ad8bMapping the Reaction Coordinates of Enzymatic DefluorinationChan, Peter W. Y.; Yakunin, Alexander F.; Edwards, Elizabeth A.; Pai, Emil F.Journal of the American Chemical Society (2011), 133 (19), 7461-7468CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The carbon-fluorine bond is the strongest covalent bond in org. chem., yet fluoroacetate dehalogenases can readily hydrolyze this bond under mild physiol. conditions. Elucidating the mol. basis of this rare biocatalytic activity will provide the fundamental chem. insights into how this formidable feat is achieved. Here, we present a series of high-resoln. (1.15-1.80 Å) crystal structures of a fluoroacetate dehalogenase, capturing snapshots along the defluorination reaction: the free enzyme, enzyme-fluoroacetate Michaelis complex, glycolyl-enzyme covalent intermediate, and enzyme-product complex. We demonstrate that enzymic defluorination requires a halide pocket that not only supplies three hydrogen bonds to stabilize the fluoride ion but also is finely tailored for the smaller fluorine halogen atom to establish selectivity toward fluorinated substrates. We have further uncovered dynamics near the active site which may play pivotal roles in enzymic defluorination. These findings may ultimately lead to the development of novel defluorinases that will enable the biotransformation of more complex fluorinated org. compds., which in turn will assist the synthesis, detoxification, biodegrdn., disposal, recycling, and regulatory strategies for the growing markets of organofluorines across major industrial sectors.97Miranda-Rojas, S.; Toro-Labbe, A. Mechanistic insights into the dehalogenation reaction of fluoroacetate/fluoroacetic acid. J. Chem. Phys. 2015, 142, 194301, DOI: 10.1063/1.4920946[Crossref], [PubMed], [CAS], Google Scholar97https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXoslOrtLc%253D&md5=e335eff76c2b5edf972eb445c484d5c9Mechanistic insights into the dehalogenation reaction of fluoroacetate/fluoroacetic acidMiranda-Rojas, Sebastian; Toro-Labbe, AlejandroJournal of Chemical Physics (2015), 142 (19), 194301/1-194301/9CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)Fluoroacetate is a toxic compd. whose environmental accumulation may represent an important contamination problem, its elimination is therefore a challenging issue. Fluoroacetate dehalogenase catalyzes its degrdn. through a two step process initiated by an SN2 reaction in which the aspartate residue performs a nucleophilic attack on the carbon bonded to the fluorine; the second step is hydrolysis that releases the product as glycolate. In this paper, we present a study based on d. functional theory calcns. of the SN2 initiation reaction modeled through the interaction between the substrate and the propionate anion as the nucleophile. Results are analyzed within the framework of the reaction force and using the reaction electronic flux to identify and characterize the electronic activity that drives the reaction. Our results reveal that the selective protonation of the substrate catalyzes the reaction by decreasing the resistance of the structural and electronic reorganization needed to reach the transition state. Finally, the reaction energy is modulated by the degree of stabilization of the fluoride anion formed after the SN2 reaction. In this way, a site-induced partial protonation acts as a chem. switch in a key process that dets. the output of the reaction. (c) 2015 American Institute of Physics.98Li, Y.; Zhang, R.; Du, L.; Zhang, Q.; Wang, W. Catalytic mechanism of C–F bond cleavage: insights from QM/MM analysis of fluoroacetate dehalogenase. Catal. Sci. Technol. 2016, 6, 73– 80, DOI: 10.1039/C5CY00777A[Crossref], [CAS], Google Scholar98https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFShtr3E&md5=ad0a17050e722c1e117dd21015fbac23Catalytic mechanism of C-F bond cleavage: insights from QM/MM analysis of fluoroacetate dehalogenaseLi, Yanwei; Zhang, Ruiming; Du, Likai; Zhang, Qingzhu; Wang, WenxingCatalysis Science & Technology (2016), 6 (1), 73-80CODEN: CSTAGD; ISSN:2044-4753. (Royal Society of Chemistry)The catalytic mechanisms of fluoroacetate dehalogenase (FAcD) toward substrates fluoroacetate and chloroacetate were studied by a combined quantum mechanics/mol. mechanics (QM/MM) method. There are twenty snapshots considered for each of the three individual systems. By analyzing multiple independent snapshots, pos. or neg. relationships between energy barriers and structural parameters in defluorination and dechlorination processes were established. We have also shown that conformational variations may cause enzymic preference differences toward competitive pathways. Besides residues Arg111, Arg114, His155, Trp156, and Tyr219, the importance of residues His109, Asp134, Lys181, and His280 during the defluorination process were also highlighted through electrostatic anal. These results may provide clues for designing new biomimetic catalysts toward degrdn. of fluorinated compds.99Wang, J. B.; Ilie, A.; Yuan, S.; Reetz, M. T. Investigating substrate scope and enantioselectivity of a defluorinase by a stereochemical probe. J. Am. Chem. Soc. 2017, 139, 11241– 11247, DOI: 10.1021/jacs.7b06019[ACS Full Text], [CAS], Google Scholar99https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1eks7rE&md5=ad6fc8676e19c3964d6ccf9426527ab1Investigating Substrate Scope and Enantioselectivity of a Defluorinase by a Stereochemical ProbeWang, Jian-bo; Ilie, Adriana; Yuan, Shuguang; Reetz, Manfred T.Journal of the American Chemical Society (2017), 139 (32), 11241-11247CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The possibility of a double Walden inversion mechanism of the defluorinase, fluoroacetate dehalogenase FAcD (RPA1163), was studied by subjecting rac-2-fluoro-2-phenylacetic acid to the defluorination process. This stereochem. probe led to inversion of configuration in a kinetic resoln. with an extremely high selectivity factor (E = >500), showing that the classical mechanism involving SN2 reaction by Asp-110 pertained. The high preference for the (S)-substrate was of synthetic value. The wide substrate scope of RPA1163 in such hydrolytic kinetic resolns. can be expected because the reaction of the even more sterically demanding rac-2-fluoro-2-benzylacetic acid proceeded similarly. Substrate acceptance and stereoselectivity were explained by extensive mol. modeling and mol. dynamics simulation computations. These computations were also applied to fluoroacetic acid itself, leading to further insights.100Tong, Z.; Board, P. G.; Anders, M. W. Glutathione transferase zeta-catalyzed biotransformation of dichloroacetic acid and other α-haloacids. Chem. Res. Toxicol. 1998, 11, 1332– 1338, DOI: 10.1021/tx980144f[ACS Full Text], [CAS], Google Scholar100https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXmsVWjtLk%253D&md5=03d205a0bf95e6836799c6321893417dGlutathione Transferase Zeta-Catalyzed Biotransformation of Dichloroacetic Acid and Other α-HaloacidsTong, Zeen; Board, Philip G.; Anders, M. W.Chemical Research in Toxicology (1998), 11 (11), 1332-1338CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)Dichloroacetic acid (DCA) is a common drinking-water contaminant, is hepatocarcinogenic in rats and mice, and is a therapeutic agent used clin. in the management of lactic acidosis. Recent studies show that glutathione transferase Zeta (GSTZ) catalyzes the oxygenation of DCA to glyoxylic acid (1998). In the present studies, the substrate selectivity of GSTZ, the kinetics of DCA metab., and the fate of DCA and glutathione were investigated. The results showed that GSTZ catalyzed the oxygenation of bromochloro-, bromofluoro-, chlorofluoro-, dibromo-, and dichloroacetic acid, but not difluoroacetic acid, to glyoxylic acid. GSTZ also catalyzed the biotransformation of fluoroacetic acid to S-(carboxymethyl)glutathione, and of (R,S)-2-bromopropionic acid, (R)-, (S)-, and (R,S)-2-chloropropionic acid, and (R,S)-2-iodopropionic acid, but not (R,S)-2-fluoropropionic acid, to S-(α-methylcarboxymethyl)glutathione; and of 2,2-dichloropropionic acid to pyruvate. No biotransformation of 3,3-dichloropropionic acid was detected, and no GSTZ-catalyzed fluoride release from Et fluoroacetate and fluoroacetamide was obsd. The relative rates of DCA biotransformation by hepatic cytosol were mouse > rat > human. Immunoblotting showed the presence of GSTZ in mouse, rat, and human liver cytosol. 13C NMR spectroscopic studies showed that [2-13C]glyoxylic acid was the only observable, stable metabolite of [2-13C]DCA. Also, glutathione was required, but was neither consumed nor oxidized to glutathione disulfide, during the oxygenation of DCA to glyoxylic acid. These results are consistent with a reaction mechanism that involves displacement of chloride from DCA by glutathione to afford S-(α-chlorocarboxymethyl)glutathione, which may undergo hydrolysis to give the hemithioacetal S-(α-hydroxycarboxymethyl)glutathione. Elimination of glutathione from the hemithioacetal would give glyoxylic acid.101Soiefer, A. I.; Kostyniak, P. J. Purification of a fluoroacetate-specific defluorinase from mouse liver cytosol. J. Biol. Chem. 1984, 259, 10787– 10792[PubMed], [CAS], Google Scholar101https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXmt1Ggu7k%253D&md5=5202bbd98e7f723aab7dd5950389ecf7Purification of a fluoroacetate-specific defluorinase from mouse liver cytosolSoiefer, Andrew I.; Kostyniak, Paul J.Journal of Biological Chemistry (1984), 259 (17), 10787-92CODEN: JBCHA3; ISSN:0021-9258.Fluoroacetate-specific defluorinase (I), an enzyme which catalyzes the release of F- from the rodenticide, fluoroacetate, was purified 347-fold from mouse liver cytosol and shown to be distinct from multiple cationic and anionic glutathione S-transferase isoenzymes. Fluoroacetate-specific I was obtained at a final specific activity of 659 nmol of F-/min/mg protein and was prepd. in an overall yield of 12%. The pI of this hepatic enzyme was acidic, at pH 6.4, as detd. by column chromatofocusing. The mol. wt. of the active species was estd. at 41,000 and SDS-polyacrylamide gels of purified I demonstrated a predominant subunit of mol. wt. 27,000. Chromatofocusing completely partitioned fluoroacetate-specific I from 2 sep. peaks of murine anionic glutathione S-transferase activity. Rabbit antibodies prepd. against purified hepatic I quant. pptd. native I from mouse and rat liver, but were unable to immunoppt. cationic or anionic glutathione S-transferase enzymes from the same prepn. The evidence presented suggests that fluoroacetate-specific I and glutathione S-transferase activities are catalyzed by sep. proteins present in the cytosol of mouse liver.102Kostyniak, P. J.; Soiefer, A. I. The role of fluoroacetate-specific dehalogenase and glutathione transferase in the metabolism of fluoroacetamide and 2,4-dinitrofluorobenzene. Toxicol. Lett. 1984, 22, 217– 222, DOI: 10.1016/0378-4274(84)90069-9[Crossref], [PubMed], [CAS], Google Scholar102https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXmt1Ciuro%253D&md5=e4eafada4752417c0223d81d968c16acThe role of fluoroacetate-specific dehalogenase and glutathione transferase in the metabolism of fluoroacetamide and 2,4-dinitrofluorobenzeneKostyniak, P. J.; Soiefer, A. I.Toxicology Letters (1984), 22 (2), 217-22CODEN: TOLED5; ISSN:0378-4274.2,4-Dinitrofluorobenzene (DNFB)(I) [70-34-8] reacts with glutathione [70-18-8] to form a stable product similar to that formed with the model glutathione S-transferase (GST) [50812-37-8] substrate, 1-chloro-2,4-dinitrobenzene (CDNB) [97-00-7]. DNFB is ∼140 times as reactive as CDNB in this chem. reaction. The enzymic defluorination of DNFB also proceeds at a more rapid rate than CDNB in the GST assay. Fluoroacetamide (FAM) [640-19-7], like fluoroacetate (FAC) [144-49-0] undergoes no discernable chem. defluorination. Its enzymic defluorination is ∼10% of that obsd. for FAC and only 0.2% of the rate for DNFB. An antibody raised to the fluoroacetate-specific dehalogenase (FSD) [37289-40-0] pptd. FAC and FAM defluorinating activity, but had no effect on CDNB or DNFB activity. Thus, DNFB is metabolized by the GST while FAM is metabolized by the FSD.103Tecle, B.; Casida, J. E. Enzymatic defluorination and metabolism of fluoroacetate, fluoroacetamide, fluoroethanol, and (−)-erythro-fluorocitrate in rats and mice examined by 19F and 13C NMR. Chem. Res. Toxicol. 1989, 2, 429– 435, DOI: 10.1021/tx00012a012[ACS Full Text], [CAS], Google Scholar103https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXmtFSgsr4%253D&md5=f38dbfdf56eb267c89a237b4c068583fEnzymatic defluorination and metabolism of fluoroacetate, fluoroacetamide, fluoroethanol, and (-)-erythro-fluorocitrate in rats and mice examined by fluorine-19 and carbon-13 NMRTecle, Berhane; Casida, John E.Chemical Research in Toxicology (1989), 2 (6), 429-35CODEN: CRTOEC; ISSN:0893-228X.Fluoroacetate administered i.p. to rats and mice is defluorinated to give fluoride ion evident in urine and kidney by 19F NMR. The use of [2-13C]- and [1,2-14C]fluoroacetate, prepd. from isotopically labeled glycine, combined with 13C NMR and TLC radioautog., resp., reveals a complex mixt. of urinary metabolites including an S-(carboxymethyl) conjugate complex in rats and mice and sulfoxidn. products thereof in rats. Direct 13C NMR examn. of the bile following treatment with [2-13C]fluoroacetate shows the presence of S-(carboxymethyl)glutathione or a related conjugate and an O-conjugate of fluoroacetate. Incubation of [13C]fluoroacetate with rat and mouse liver cytosol involves formation of S-[([13C]carboxymethyl)glutathione and fluoride ion. Fluorocitrate is also detected by 19F NMR examn. of fluoroacetate incubations with mouse liver cytosol. Fluoroacetamide administered i.p. to rats and mice yields urinary fluoride ion formed via fluoroacetate which is liberated on hydrolysis by an organophosphate-sensitive amidase. 19F NMR chem. shifts of other metabolites of fluoroacetamide are consistent with fluoroacetohydroxamic acid in the liver of mice and fluorocitrate in the urine of rats. Fluoroethanol gives urinary fluoroacetate and fluoride ion in rats and mice and is converted to fluoroacetaldehyde by mouse and rat liver microsomes. (-) And (+)-erythro-fluorocitrates administered i.p. to rats yield mostly the parent compds. in urine at 6 h with increasing amts. of fluoride ion thereafter. 19F NMR establishes that rat and mouse liver cytosols defluorinate (-)-, but not (+)-erythro-fluorocitrate and pig heart aconitase also defluorinates (-)-erythro-fluorocitrate. Metabolic defluorination of fluoroacetate and its progenitors, fluoroacetamide and fluoroethanol, is therefore attributable to both conjugation of fluoroacetate with GSH and conversion to (-)-erythro-fluorocitrate, which is both an inhibitor of and a substrate for aconitase. 13C NMR spectra of urine of rats and mice poisoned with fluoroacetate or (-)-erythro-fluorocitrate show elevated citrate and glucose and diminished urea consistent with disruptions in the tricarboxylic acid cycle and NH3 metab.104Kim, K.-H.; Shon, Z.-H.; Nguyen, H. T.; Jeon, E.-C. A review of major chlorofluorocarbons and their halocarbon alternatives in the air. Atmos. Environ. 2011, 45, 1369– 1382, DOI: 10.1016/j.atmosenv.2010.12.029[Crossref], [CAS], Google Scholar104https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFeis7Y%253D&md5=2e83d2921958c8033e2f28df1934949eA review of major chlorofluorocarbons and their halocarbon alternatives in the airKim, Ki-Hyun; Shon, Zang-Ho; Nguyen, Hang Thi; Jeon, Eui-ChanAtmospheric Environment (2011), 45 (7), 1369-1382CODEN: AENVEQ; ISSN:1352-2310. (Elsevier Ltd.)A review; to establish a proper regulation strategy on the emissions of major halocarbons including chlorofluorocarbons (CFCs), carbon tetrachloride (CCl4), hydrochlorofluorocarbons (HCFCs) and hydrofluorocarbons (HFCs), it is essential to assess their environmental behavior in relation to social and technol. changes for their control. The prodn. and release of CFCs and CCl4 increased rapidly to peak between the 1970s and 1980s and then decreased dramatically from the end of the 1980s in good accordance with the phase out schedules set by the Montreal Protocol. Both HCFCs (e.g., HCFC-124, HCFC-141b, and HCFC-142b) and HFCs (e.g., HFC-134a) have been introduced as CFCs alternatives between the late 1980s and early 1990s. However, these alternatives have already been or will be scheduled to be phased out because of their involvement in ozone destruction and global warming. In light of all the complexities assocd. with the global chem. of CFCs and their alternatives, this paper provides an overview of their prodn. and emission trends, their relationship with the ozone depletion phenomenon, the chem. regulating their removal processes, and their distribution patterns with diverse temporal and spatial scales.105Anders, M. W. Metabolism and toxicity of hydrochlorofluorocarbons: current knowledge and needs for the future. Environ. Health Perspect. 1991, 96, 185– 191, DOI: 10.1289/ehp.9196185[Crossref], [PubMed], [CAS], Google Scholar105https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xkt1Wms7k%253D&md5=d2f4f940f2d5464ed79764e06a8059cbMetabolism and toxicity of hydrochlorofluorocarbons: current knowledge and needs for the futureAnders, M. W.Environmental Health Perspectives (1991), 96 (), 185-91CODEN: EVHPAZ; ISSN:0091-6765.A review with 63 refs. Hydrochlorofluorocarbons (HCFCs) are being developed as replacements for chlorofluorocarbons (CFCs) that deplete stratospheric ozone. The depletion of stratospheric ozone may increase the intensity of UV radiation at the earth's surface, which may be assocd. with global, adverse human health effects. The greater tropospheric lability of HCFCs, which is due to the presence of C-H bonds, reduces HCFC migration to the stratosphere; HCFCs should, therefore, cause less depletion of stratospheric ozone than CFCs. HCFCs under development include HCFC-22 (chlorodifluoromethane), HCFC-123 (2,2-dichloro-1,1,1-trifluoroethane), HCFC-132b (1,2-dichloro-1,1-difluoroethane), HCFC-134a (1,1,1,2-tetrafluoroethane), HCFC-141b (1,1-dichloro-1-fluoroethane, and HCFC-142b (1-chloro-1,1-difluoroethane). With the exception of HCFC-22, which is already in use, the metab. and toxicity of HCFCs have not been studied in detail. By analogy to chlorinated ethanes, predictions can be made about the possible metab. of HCFCs, but there are insufficient data available to predict rates of metab. Although most HCFCs appear to show low acute toxicity, some HCFCs are mutagenic in the Ames test. Hence, future research on HCFCs should include studies on the in vivo and in vitro metab. of HCFCs as well as on their toxicity in in vivo and in vitro systems.106Urban, G.; Dekant, W. Metabolism of 1,1-dichloro-2,2,2-trifluoroethane in rats. Xenobiotica 1994, 24, 881– 892, DOI: 10.3109/00498259409043287[Crossref], [PubMed], [CAS], Google Scholar106https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXmsVynsLw%253D&md5=42c9c0f6a5266d7ca1de4b1947502296Metabolism of 1,1-dichloro-2,2,2-trifluoroethane in ratsUrban, G.; Dekant, W.Xenobiotica (1994), 24 (9), 881-92CODEN: XENOBH; ISSN:0049-8254.1,1-Dichloro-2,2,2-trifluoro-[2-14C]-ethane (HCFC-123) is a chlorofluorohydrocarbon with potential widespread use and assocd. human exposure. As a part of the toxicol. evaluation of HCFC-123, its metab. was studied in rodents in a closed recirculating exposure system. Two male rats were individually exposed for 6 h. Excretion of radioactivity was monitored for 48 h after the start of the exposure. Of the radioactivity introduced into the chamber, 14% was recovered in urine within the period of observation. Excretion of metabolites in the urine was very slow. Trifluoroacetic acid was the major metabolite of HCFC-123 and N-trifluoroacetyl-2-aminoethanol and N-acetyl-S-(2,2-dichloro-1,1-difluoroethyl)-L-cysteine were identified as minor urinary metabolites of HCFC-123. Forty-eight hours after the start of the exposure, covalent binding of radioactive metabolites to protein was highest in liver followed by kidney and lung. Covalent binding above background levels was not obsd. in pancreas and testis, the target organs of HCFC-123 tumorigenicity. These results suggest that the biotransformation of HCFC-123 in rodents follows a pathway identical to those of the extensively studied structural analog halothane.107Urban, G.; Speerschneider, P.; Dekant, W. Metabolism of the chlorofluorocarbon substitute 1,1-dichloro-2,2,2-trifluoroethane by rat and human liver microsomes: the role of cytochrome P450 2E1. Chem. Res. Toxicol. 1994, 7, 170– 176, DOI: 10.1021/tx00038a009[ACS Full Text], [CAS], Google Scholar107https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXhvVCrsL8%253D&md5=e59b32f3e2b9891d2c6df95d05e5201aMetabolism of the Chlorofluorocarbon Substitute 1,1-Dichloro-2,2,2-trifluoroethane by Rat and Human Liver Microsomes: The Role of Cytochrome P450 2E1Urban, Gudrun; Speerschneider, Petra; Dekant, WolfgangChemical Research in Toxicology (1994), 7 (2), 170-6CODEN: CRTOEC; ISSN:0893-228X.1,1-Dichloro-2,2,2-trifluoroethane (HCFC-123) has been developed as a substitute for ozone-depleting chlorofluorocarbons. The authors compared the metab. of HCFC-123 and its analog halothane in rat and human liver microsomes. 19F-NMR studies showed that trifluoroacetic acid is a major metabolite of HCFC-123. Besides trifluoroacetic acid, chlorodifluoroacetic acid and inorg. fluoride were identified as products of the enzymic oxidn. of HCFC-123 in rat and human liver microsomes by 19F-NMR and mass spectrometry. These metabolites were not detected in incubations with halothane. HCFC-123 and halothane were transformed by liver microsomes from untreated rats at low rates. Microsomes from ethanol- and pyridine-treated rats metabolized both HCFC-123 and halothane at much higher rates. These microsomes also exhibited high rates of p-nitrophenol oxidn. P-Nitrophenol is a model substrate mainly oxidized by P 450 2E1 to p-nitrocatechol. Samples of human liver microsomes showed considerable differences in the extent of HCFC-123, p-nitrophenol oxidn., and chlorzoxazone hydroxylation. In human liver microsomes, rabbit anti-rat P 450 2E1 IgG recognized a single protein band corresponding in apparent mol. wt. to human P 450 2E1. Immunoblot anal. revealed considerable heterogenity in the P 450 2E1 protein content of the human liver samples. Trifluoroacetic acid formation from HCFC-123 and halothane and p-nitrocatechol formation from p-nitrophenol were significantly reduced by the P 450 2E1 inhibitor diethyldithiocarbamate. P-Nitrophenol also inhibited halothane and HCFC-123 oxidn. in both rat and human liver microsomes. Moreover, the rates of trifluoroacetic acid formation from HCFC-123 and halothane correlated well with the ability of rat and human liver microsomes to oxidize p-nitrophenol and chlorzoxazone and the amt. of P 450 2E1 protein in liver microsomes detd. by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot anal. These data indicate that cytochrome P 450 2E1 plays a major role in the metab. of HCFC-123 and halothane in vitro.108Olson, M. J.; Johnson, J. T.; O’Gara, J. F.; Surbrook, S. E., Jr. Metabolism in vivo and in vitro of the refrigerant substitute 1,1,1,2-tetrafluoro-2-chloroethane. Drug Metab. Dispos. 1991, 19, 1004– 1011[PubMed], [CAS], Google Scholar108https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXmt1Glu7c%253D&md5=edf8bcea5ee06045607c6e6c47a36383Metabolism in vivo and in vitro of the refrigerant substitute 1,1,1,2-tetrafluoro-2-chloroethaneOlson, Michael J.; Johnson, John T.; O'Gara, John F.; Surbrook, Stephen E., Jr.Drug Metabolism and Disposition (1991), 19 (5), 1004-11CODEN: DMDSAI; ISSN:0090-9556.Ternary mixts. of hydrochlorofluorocarbons and hydrofluorocarbons are being evaluated as refrigerant substitutes for dichlorodifluoromethane, which is to be banned from further prodn. in 2000. A priori consideration of the similarity between 1,1,1,2-tetrafluoro-2-chloroethane (HCFC-124), a primary component of candidate refrigerant blends, and halothane suggests that metab. of HCFC-124 might proceed via reactive intermediates. The data show that rats exposed for 2 h to ∼10,000 ppm HCFC-124 excreted both inorg. fluoride (F-) and trifluoroacetic acid (TFA), identified by 9F-NMR, in the urine. Likewise, microsomes produced F- and TFA from HCFC-124 in an NADPH-dependent, CO-inhibited, aerobic reaction. Treatment of rats with pyridine caused about a 20-fold increase in aerobic microsomal metab. (F- release) of HCFC-124, while the rate of defluorination was slightly decreased by phenobarbital administration. An antibody to cytochrome P 450 IIE1 inhibited more than 90% of HCFC-124 metab. in pyridine-induced prepns. Defluorination of HCFC-124 by microsomes also occurred under conditions of greatly reduced oxygen tension, demonstrating that this halocarbon can be reductively metabolized. Moreover, heat-inactivated, NADPH-reduced microsomes liberated F- and a fluorinated org. product, although not TFA, from HCFC-124. Formation of TFA and F- as products of oxidative HCFC-124 metab. support the hypothesis that trifluoroacetyl fluoride is formed as an intermediate. Trifluoroacetyl halides are known to adduct tissue proteins. The reductive metab. of HCFC-124, by analogy to halothane, may produce a radical (•CHFCF) capable of biol. interactions. Although the authors offer evidence for the cytochrome P 450-dependent metab. of HCFC-124 both in vivo and in vitro, it is, at present, unknown whether the rate of HCFC-124 metab. in vivo is sufficient to result in trifluoroacetylation of tissue proteins or other protoxic events mediated by reactive intermediates.109Yin, H.; Jones, J. P.; Anders, M. W. Metabolism of 1-fluoro-1,1,2-trichloroethane, 1,2-dichloro-1,1-difluoroethane, and 1,1,1-trifluoro-2-chloroethane. Chem. Res. Toxicol. 1995, 8, 262– 268, DOI: 10.1021/tx00044a012[ACS Full Text], [CAS], Google Scholar109https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXjvVSksrk%253D&md5=f832e3f587d90005f9736302d2dfeddaMetabolism of 1-Fluoro-1,1,2-trichloroethane, 1,2-Dichloro-1,1-difluoroethane, and 1,1,1-Trifluoro-2-chloroethaneYin, Hequn; Jones, Jeffrey P.; Anders, M. W.Chemical Research in Toxicology (1995), 8 (2), 262-8CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)1-Fluoro-1,1,2-trichloroethane (HCFC-131a), 1,2-dichloro-1,1-difluoroethane (HCFC-132b), and 1,1,1-trifluoro-2-chloroethane (HCFC-133a) were chosen as models for comparative metab. studies on 1,1,1,2-tetrahaloethanes, which are under consideration as replacements for ozone-depleting chlorofluorocarbons (CFCs). Male Fischer 344 rats were given 10 mmol/kg i.p. HCFC-131a or HCFC-132b or exposed by inhalation to 1% HCFC-133a for 2 h. Urine collected in the first 24 h after exposure was analyzed by 19F NMR and GC/MS and with a fluoride-selective ion electrode for the formation of fluorine-contg. metabolites. Metabolites of HCFC-131a included 2,2-dichloro-2-fluoroethyl glucuronide, 2,2-dichloro-2-fluoroethyl sulfate, dichlorofluoroacetic acid, and inorg. fluoride. Metabolites of HCFC-132b were characterized as 2-chloro-2,2-difluoroethyl glucuronide, 2-chloro-2,2-difluoroethyl sulfate, chlorodifluoroacetic acid, chlorodifluoroacetaldehyde hydrate, chlorodifluoroacetaldehyde-urea adduct, and inorg. fluoride. HCFC-133a was metabolized to 2,2,2-trifluoroethyl glucuronide, trifluoroacetic acid, trifluoroacetaldehyde hydrate, trifluoroacetaldehyde-urea adduct, inorg. fluoride, and a minor, unidentified metabolite. With HCFC-131a and HCFC-132b, glucuronide conjugates of 2,2,2-trihaloethanols were the major urinary metabolites, whereas with HCFC-133a, a trifluoroacetaldehyde-urea adduct was the major urinary metabolite. Anal. of metabolite distribution in vivo indicated that aldehydic metabolites increased as fluorine substitution increased in the order HCFC-131a < HCFC-132b < HCFC-133a. With NADPH-fortified rat liver microsomes, HCFC-133a and HCFC-132b were biotransformed to trifluoroacetaldehyde and chlorodifluoroacetaldehyde, resp., whereas HCFC-131a was converted to dichlorofluoroacetic acid. No covalently bound metabolites of HCFC-131a and HCFC-133a metabolites were detected by 19F NMR spectroscopy. The nature of the identified org. fluorine-contg. metabolites indicates that cytochrome P 450-dependent oxidn. predominates in the metab. of these 1,1,1,2-tetrahaloethanes. The generation of fluoride from the fluorodihalomethyl group (-CFX2) apparently arises from a sep. dehalogenation pathway.110Harris, J. W.; Anders, M. W. Metabolism of the hydrochlorofluorocarbon 1,2-dichloro-1,1-difluoroethane. Chem. Res. Toxicol. 1991, 4, 180– 186, DOI: 10.1021/tx00020a009[ACS Full Text], [CAS], Google Scholar110https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXhtFKmsrc%253D&md5=6aaa6391e895b4178d6acc9baf150325Metabolism of the hydrochlorofluorocarbon 1,2-dichloro-1,1-difluoroethaneHarris, James W.; Anders, M. W.Chemical Research in Toxicology (1991), 4 (2), 180-6CODEN: CRTOEC; ISSN:0893-228X.Male rats were given 10 mmol/kg 1,2-dichloro-1,1-difluoroethane (HCFC-132b) dissolved in corn oil by i.p. injection. An NMR assay for covalent binding of HCFC-132b metabolites to liver proteins was neg., whereas binding was obsd. in halothane-treated rats. Total urinary metabolites excreted by rats given HCFC-132b during the first 24 h amounted to 1.8% of the injected dose, as detd. by 19F NMR. During the first 6 h, metabolites of HCFC-132b corresponding to 2-chloro-2,2-difluoroethyl glucuronide, unknown metabolite A, chlorodifluoroacetic acid, and chlorodifluoroacetaldehyde hydrate [both free and conjugated (unknown metabolite B)] were excreted in urine in the approx. ratio 100 : 9 : 3 : 7, resp. Metabolite A is apparently an O-conjugate of 2-chloro-2,2-difluoroethanol; unconjugated 2-chloro-2,2-difluoroethanol was not detected in urine. The 19F NMR spectrum of metabolite B indicates the formation of a hemiacetal of chlorodifluoroacetaldehyde. Repeated exposure of rats to HCFC-132b significantly increased both the rate of chlorodifluoroacetic acid excretion and the relative fraction of the HCFC-132b dose excreted as chlorodifluoroacetic acid in urine. Incubation of HCFC-132b with rat hepatic microsomes yielded chlorodifluoroacetaldehyde hydrate as the only fluorinated product. The in vitro metab. of HCFC-132b was increased in microsomes from pyridine-treated rats as compared with control rats and HCFC-132b metab. was inhibited by p-nitrophenol, indicating that the cytochrome P 450 isoform IIE1 is largely responsible for the initial hydroxylation of HCFC-132b.111Rusch, G. M.; Coombs, D.; Hardy, C. The acute, genetic, developmental, and inhalation toxicology of 1,1,1,3,3-pentafluoropropane (HFC 245fa). Toxicol. Sci. 1999, 52, 289– 301, DOI: 10.1093/toxsci/52.2.289[Crossref], [PubMed], [CAS], Google Scholar111https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXhtF2itA%253D%253D&md5=0955f74d095eb9344c1ebf0ab27f935cThe acute, genetic, developmental, and inhalation toxicology of 1,1,1,3,3-pentafluoropropane (HFC 245fa)Rusch, George M.; Coombs, Derek; Hardy, ColinToxicological Sciences (1999), 52 (2), 289-301CODEN: TOSCF2; ISSN:1096-6080. (Oxford University Press)1,1,1,3,3-Pentafluoropropane (HFC 245fa) is a volatile, low boiling liq. Tt was inactive in a reverse mutation (Ames) assay using 5 strains of Salmonella typhimurium and 1 strain of Escherichia coli. It was also inactive in an in vivo mouse micronucleus assay with exposures of 101,000 ppm. In a chromosome aberration study with human lymphocytes, some activity was seen when cell cultures were exposed to atmospheres of 30% vol./vol. or higher for 24 h without metabolic activation. No activity was seen in assays using <30% vol./vol. or exposure times of less than 24 h. No activity was seen in the presence of metabolic activation even with exposures of 70%. It was not toxic by the dermal route. There was no mortality or significant signs of toxicity when rats and mice were given 4 h exposures to levels of 203,000 ppm or 101,000 ppm of HFC 245fa, resp. In a cardiac sensitization study with dogs involving i.v. administration of epinephrine, the no obsd. effect level (NOEL) was 34,000 ppm and the threshold for a response was 44,000 ppm. In a rat inhalation, developmental toxicity study, a slight redn. in pup wt. was seen at 50,000 ppm, but not at 10,000 ppm. There were no developmental effects at any level. A series of 3 inhalation toxicity studies were conducted. All involved daily 6 h exposures ≤50,000 ppm. The 1st study involved 14 consecutive snout-only exposures. There were no treatment-related effects on body wt., survival, or histol. parameters. BUN, GPT, and GOT levels frequently were elevated compared to controls, whereas cholesterol levels tended to be lower. The 2nd study involved 28 consecutive whole-body exposures. Again, there were no treatment related effects on body wt., survival, or histol. parameters. Urine vol. was increased. Increases were also seen in several red blood cell parameters. These may be related to partial dehydration. Increases were seen in BUN levels and alk. phosphatase (AP), GPT, GOT, and CPK activities, primarily in rats exposed at 10,000 and 50,000 ppm. Urinary fluoride levels were also elevated in an exposure- related pattern. In the 3rd study, whole-body exposures were conducted 5 days per wk for 13 wk. There were no treatment-related effects on survival, clin. observations, body wt. gain, or food consumption. Urine vols. were increased, urinary fluoride levels were elevated, and increases were seen in red blood cell counts, and related parameters and increases were seen in AP, GOT, GPT, and CPK activities. These effects were seen in the 10,000 and 50,000 ppm exposure level groups. Histopathol. examn. did not show any effects on the kidney, liver, or lungs. There was an increased incidence of myocarditis in all animals exposed at 50,000 ppm and the majority exposed at 10,000 ppm. It was described as mild. Based on these findings, 2000 ppm appears to be a no obsd. adverse effect level.112Bayer, T.; Amberg, A.; Bertermann, R.; Rusch, G. M.; Anders, M. W.; Dekant, W. Biotransformation of 1,1,1,3,3-pentafluoropropane (HFC-245fa). Chem. Res. Toxicol. 2002, 15, 723– 733, DOI: 10.1021/tx025505c[ACS Full Text], [CAS], Google Scholar112https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XivFWitb0%253D&md5=9fb4489e1de801b3cb9a6183168312d9Biotransformation of 1,1,1,3,3-Pentafluoropropane (HFC-245fa)Bayer, Tanja; Amberg, Alexander; Bertermann, Ruediger; Rusch, George M.; Anders, M. W.; Dekant, WolfgangChemical Research in Toxicology (2002), 15 (5), 723-733CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)1,1,1,3,3-Pentafluoropropane (HFC-245fa) is being developed as a CFC substitute. 1,1,1,3,3-Pentafluoropropane has a low potential for toxicity: the only remarkable toxic effect seen in rats after inhalation exposure to 1,1,1,3,3-pentafluoropropane in concns. of up to 50,000 ppm for 90 days was an increased incidence of diffuse myocarditis. To elucidate the possible role of biotransformation in 1,1,1,3,3-pentafluoropropane-induced cardiotoxicity, the biotransformation of 1,1,1,3,3-pentafluoropropane was investigated in rats after inhalation exposure and in rat and human liver microsomes. Male and female rats were exposed by inhalation to 50 000, 10 000, and 2000 ppm 1,1,1,3,3-pentafluoropropane for 6 h, urine was collected for 72 h, and metabolites excreted were identified by 19F NMR spectroscopy and quantified by GC/MS. Trifluoroacetic acid and inorg. fluoride were identified as major urinary metabolites of 1,1,1,3,3-pentafluoropropane; 3,3,3-trifluoropropanoic acid and 1,1,1,3,3-pentafluoropropane-2-ol were minor metabolites. The extent of 1,1,1,3,3-pentafluoropropane biotransformation after inhalation was dependent on exposure concns. Neither 3,3,3-trifluoropropanoic acid nor 3,3,3-trifluoropyruvic acid were metabolized to trifluoroacetic acid in vitro or in rats. In rat and human liver microsomes, 1,1,1,3,3-pentafluoropropane was biotransformed by a cytochrome P 450-dependent reaction to trifluoroacetic acid and 3,3,3-trifluoropropanoic acid. Rates of trifluoroacetic acid formation were 99.2±20.5 pmol (mg of protein)-1 min-1 and of 3,3,3-trifluoropropanoic acid formation were 17.5±4.0 pmol (mg of protein)-1 min-1 in liver microsomes from male rats. In human liver microsomes, rates of trifluoroacetic acid formation ranged from 0 to 11.6 pmol (mg of protein)-1 min-1, and rates of 3,3,3-trifluoropropanoic acid formation ranged from 0.7 to 7.6 pmol (mg of protein)-1 min-1. The results show that 1,1,1,3,3-pentafluoropropane is metabolized at low rates in vivo and in vitro. The toxic effects of 1,1,1,3,3-pentafluoropropane may be assocd. with the formation of the minor metabolite 3,3,3-trifluoropropanoic acid, which is highly toxic in rats.113Mitsch, R. A.; Robertson, J. E. Difluorodiazirine. V. Difluoromethyl esters and ethers. J. Heterocycl. Chem. 1965, 2, 152– 156, DOI: 10.1002/jhet.5570020209[Crossref], [CAS], Google Scholar113https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF2MXktlWntr4%253D&md5=1188bac34dd03f3018f85f4a45745af3Difluorodiazirine. V. Difluoromethyl esters and ethersMitsch, Ronald A.; Robertson, Jerry E.Journal of Heterocyclic Chemistry (1965), 2 (2), 152-6CODEN: JHTCAD; ISSN:0022-152X.cf. CA 62, 13055h. The preparation of difluoromethyl esters and ethers by the photolytic reaction of difluorodiazirine with acids and alcohols, respectively, is described. Difluoromethyl benzoate is converted, under mild conditions, to methyl benzoate, benzamide, and benzanilide by reaction with methanol, ammonium hydroxide, and aniline, respectively. The difluoromethyl esters arc activated to nucleophilic reaction by virtue of the α-fluorine substitution.114Neuberger, J.; Mieli-Vergani, G.; Tredger, J. M.; Davis, M.; Williams, R. Oxidative metabolism of halothane in the production of altered hepatocyte membrane antigens in acute halothane-induced hepatic necrosis. Gut 1981, 22, 669– 672, DOI: 10.1136/gut.22.8.669[Crossref], [PubMed], [CAS], Google Scholar114https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38Xmt1Giug%253D%253D&md5=44107db5a0287cd29c095fa2ad711e4aOxidative metabolism of halothane in the production of altered hepatocyte membrane antigens in acute halothane-induced hepatic necrosisNeuberger, J.; Mieli-Vergani, Giorgina; Tredger, J. M.; Davis, M.; Williams, RogerGut (1981), 22 (8), 669-72CODEN: GUTTAK; ISSN:0017-5749.The expression of antigens on halothane [151-67-7]-altered hepatocytes was assocd. with the oxidative metab. of halothane. The susceptibility of halothane-altered cells to cytotoxicity by normal lymphocytes decreased with decreasing O tension at anesthesia. This result contrasts with those of other groups which showed that the reductive route was involved in the direct hepatotoxic reaction attributed to halothane.115McLain, G. E.; Sipes, I. G.; Brown, B. R., Jr. An animal model of halothane hepatotoxicity: roles of enzyme induction and hypoxia. Anesthesiology 1979, 51, 321– 326, DOI: 10.1097/00000542-197910000-00008[Crossref], [PubMed], [CAS], Google Scholar115https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3cXkvFU%253D&md5=97a82c2d1e394dd07137fe025f6fed5fAn animal model of halothane hepatotoxicity: roles of enzyme induction and hypoxiaMcLain, George E.; Sipes, I. Glenn; Brown, Burnell R., Jr.Anesthesiology (1979), 51 (4), 321-6CODEN: ANESAV; ISSN:0003-3022.Exposure of phenobarbital-pretreated male rats to halothane [151-67-7] (1%, for 2 h under conditions of hypoxia) resulted in extensive centrilobular necrosis within 24 h. Accompanying the morphologic damage were an increase in serum glutamic-pyruvic transaminase [9000-86-6] and a decrease in the hepatic microsomal cytochrome P-450 [9035-51-2]. Glutathione [70-18-8] levels in the liver were unchanged. Phenobarbital-pretreated rats anesthetized with halothane at higher O2 concns. had only minor morphologic changes at 24 h. Hepatic injury was not apparent in any nonphenobarbital-induced rat or in any induced animal exposed to ether [60-29-7] under hypoxic conditions. There was a 2.6-fold increase in the 24-h urinary excretion of F in those rats in which extensive centrilobular necrosis developed. The in vivo covalent binding to lipids of 14C from halothane-14C also was increased markedly when halothane-14C was administered i.p. to phenobarbital-induced rats maintained hypoxic for 2 h. Thus halothane is metabolized to hepatotoxic intermediates by a reductive or nonoxygen-dependent cytochrome P-450-dependent pathway. This animal model of halothane-induced hepatotoxicity may be clin. relevant. A decrease in hepatic blood flow during halothane anesthesia may decrease the PO2 available to hepatocytes and thus direct the metab. of halothane along its reductive, hepatotoxic pathway.116Ray, D. C.; Drummond, G. B. Halothane hepatitis. Br. J. Anaesth. 1991, 67, 84– 99, DOI: 10.1093/bja/67.1.84[Crossref], [PubMed], [CAS], Google Scholar116https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXmt1amsrg%253D&md5=b194e93271ea0709c6ed14cb1455b9c6Halothane hepatitisRay, D. C.; Drummond, G. B.British Journal of Anaesthesia (1991), 67 (1), 84-99CODEN: BJANAD; ISSN:0007-0912.A review with 187 refs.117Bourdi, M.; Chen, W.; Peter, R. M.; Martin, J. L.; Buters, J. T.; Nelson, S. D.; Pohl, L. R. Human cytochrome P450 2E1 is a major autoantigen associated with halothane hepatitis. Chem. Res. Toxicol. 1996, 9, 1159– 1166, DOI: 10.1021/tx960083q[ACS Full Text], [CAS], Google Scholar117https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XlslOksrg%253D&md5=f7e3cba9321de99ed160db4f31d00e27Human Cytochrome P450 2E1 Is a Major Autoantigen Associated with Halothane HepatitisBourdi, Mohammed; Chen, Weiqiao; Peter, Raimund M.; Martin, Jackie L.; Buters, Jeroen T. M.; Nelson, Sidney D.; Pohl, Lance R.Chemical Research in Toxicology (1996), 9 (7), 1159-1166CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)Autoantibodies against specific human cytochrome P450s have been found in the sera of patients suffering from a variety of diseases, including those caused by drugs. In the cases of tienilic acid- and dihydralazine-induced hepatitis, patients have serum autoantibodies directed against cytochromes P 450 2C9 and P 450 1A2, resp. In the present study, we have found that 25 of 56 (45%) patients diagnosed with halothane hepatitis have autoantibodies that react with human cytochrome P 450 2E1 that was purified from a baculovirus expression system. The autoantibodies inhibited the activity of cytochrome P 450 2E1 and appeared to be directed against mainly conformational epitopes. In addn., because cytochrome P 450 2E1 became trifluoroacetylated when it oxidatively metabolized halothane, it is possible that the covalently altered form of cytochrome P 450 2E1 may be able to bypass the immunol. tolerance that normally exists against cytochrome P 450 2E1. A similar mechanism may explain the formation of autoantibodies that have been found against other cellular targets of the reactive trifluoroacetyl chloride metabolite of halothane.118Kurth, M. J.; Yokoi, T.; Gershwin, M. E. Halothane-induced hepatitis: paradigm or paradox for drug-induced liver injury. Hepatology 2014, 60, 1473– 1475, DOI: 10.1002/hep.27253[Crossref], [PubMed], [CAS], Google Scholar118https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvVansbfI&md5=4d713af06e9f8ee9972a02b0d799f5dcHalothane-induced hepatitis: Paradigm or paradox for drug-induced liver injuryKurth, Mark J.; Yokoi, Tsuyoshi; Gershwin, M. EricHepatology (Hoboken, NJ, United States) (2014), 60 (5), 1473-1475CODEN: HPTLD9; ISSN:0270-9139. (John Wiley & Sons, Inc.)A review. This article describes about cellular events and signaling pathways underlying halothane-induced liver injury with emphasis on role of thymic stromal lymphopoietin receptor and cytokines such as interleukin-4 in pathogenesis in human and mouse.119Nastainczyk, W.; Ullrich, V.; Sies, H. Effect of oxygen concentration on the reaction of halothane with cytochrome P450 in liver microsomes and isolated perfused rat liver. Biochem. Pharmacol. 1978, 27, 387– 392, DOI: 10.1016/0006-2952(78)90366-0[Crossref], [PubMed], [CAS], Google Scholar119https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXkslSlt7o%253D&md5=adb0b275ee78f7481aa13072f2849e66Effect of oxygen concentration on the reaction of halothane with cytochrome P450 in liver microsomes and isolated perfused rat liverNastainczyk, Wolfgang; Ullrich, Volker; Sies, HelmutBiochemical Pharmacology (1978), 27 (4), 387-92CODEN: BCPCA6; ISSN:0006-2952.Formation of the complex between reduced cytochrome P450 [9035-51-2] and trifluoromethyl carbene [2441-28-3], formed in hepatic microsomal halothane [151-67-7] metab., was inhibited at high O concns. but began at <50μM O and was max. under anaerobic conditions. Metyrapone was an efficient inhibitor of the carbene complex formation. Organ spectrophotometry of isolated perfused livers showed that the complex appeared under slightly hypoxic conditions and that addn. of metyrapone abolished its formation. Halothane may be metabolized in vivo to reactive intermediates when the O concn. of the cell is <50μM.120Ahr, H. J.; King, L. J.; Nastainczyk, W.; Ullrich, V. The mechanism of reductive dehalogenation of halothane by liver cytochrome P450. Biochem. Pharmacol. 1982, 31, 383– 390, DOI: 10.1016/0006-2952(82)90186-1[Crossref], [PubMed], [CAS], Google Scholar120https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XktVSitbY%253D&md5=2b7cca80a26a1f6737878863cd0184aaThe mechanism of reductive dehalogenation of halothane by liver cytochrome P-450Ahr, Hans J.; King, L. J.; Nastainczyk, W.; Ullrich, V.Biochemical Pharmacology (1982), 31 (3), 383-90CODEN: BCPCA6; ISSN:0006-2952.Studies of the reductive dehalogenation of halothane (I) [151-67-7] leading to 2-chloro-1,1,1-trifluoroethane (II) [75-88-7] and 2-chloro-1,1-difluoroethylene (III) [359-10-4] in vitro in anaerobic rat liver microsomes showed that stimulation of NADPH oxidn. by I as well as formation of II and III were dependent on cytochrome P-450 (IV) [9035-51-2]. After replacement of NADPH by Na dithionite as a reducing agent, III was the only product. The product pattern was influenced by 3-methylcholanthrene, benzo[a]pyrene, phenobarbitone, and Arochlor 1254 pretreatments and by addn. of anti-IV-PB Ig. II/III ratio was shifted by addn. or inhibition of cytochrome b5 and by pH variation, indicating a crucial role of the second electron donation to IV in detg. product pattern. The intermediate complex of IV with a Soret band at 470 nm formed with I in reduced liver microsomes decompd. spontaneously to give III; the 470-nm peak may represent IV Fe3+--CHCl-CF3 carbanion complex. A reaction path was derived including radical and carbanion intermediates as reactive precursors of II and III, resp.121Baker, M. T.; Van Dyke, R. A. Reductive halothane metabolite formation and halothane binding in rat hepatic microsomes. Chem.-Biol. Interact. 1984, 49, 121– 132, DOI: 10.1016/0009-2797(84)90056-5[Crossref], [PubMed], [CAS], Google Scholar121https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXktlaku7w%253D&md5=3dd71e3ba602de20e9b1b9134c68c778Reductive halothane metabolite formation and halothane binding in rat hepatic microsomesBaker, Max T.; Van Dyke, Russell A.Chemico-Biological Interactions (1984), 49 (1-2), 121-32CODEN: CBINA8; ISSN:0009-2797.The formation of the reductive 14C-labeled halothane [151-67-7] metabolites, 2-chloro-1,1,1-trifluoroethane (CTE) [75-88-7] and 2-chloro-1,1-difluoroethylene (CDE) [359-10-4], was detd. in anaerobic microsomal incubations by HPLC. The HPLC technique used allowed accurate measurements of low levels of [14C]halothane metabolites. Comparisons of metabolic profiles and halothane binding in microsomes reduced with NADPH and Na dithionite showed that dithionite stimulated CDE prodn. and total halothane degrdn., but inhibited CTE formation and [14C]-halothane binding. Similarly, the addn. of isoflurane [26675-46-7], but not enflurane [13838-16-9], to microsomes increased CDE prodn. and decreased CTE formation and [14C]halothane-lipid binding. Measurement of F- in similar incubations show that F- release from halothane correlates with the formation of CDE and not CTE. The relative prodn. of CTE and CDE may not remain const. in microsomal prepns., and halothane binding correlates with CTE formation and not CDE and F- prodn.122Van Dyke, R. A.; Baker, M. T.; Jansson, I.; Schenkman, J. Reductive metabolism of halothane by purified cytochrome P-450. Biochem. Pharmacol. 1988, 37, 2357– 2361, DOI: 10.1016/0006-2952(88)90361-9[Crossref], [PubMed], [CAS], Google Scholar122https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXksVOrs7Y%253D&md5=f051cdde40286e63f9ad7f997a6cc5c9Reductive metabolism of halothane by purified cytochrome P-450Van Dyke, Russell A.; Baker, Max T.; Jansson, Ingela; Schenkman, JohnBiochemical Pharmacology (1988), 37 (12), 2357-61CODEN: BCPCA6; ISSN:0006-2952.The reductive metab. of halothane was detd. by using purified RLM2, PBRLM4 and PBRLM5 forms of rat liver microsomal cytochrome P 450. The metabolites, 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE), were detd. All 3 forms of cytochrome P 450 produced CTE with relatively small differences in its prodn. among the various forms. There were major differences, however, in the prodn. of CDE, with PBRLM5 being the most active. PBRLM5 was also the only form to show the development of a complex between halothane and cytochrome P 450. This complex absorbed light maximally at 470 nm. The complex formation and the prodn. of CDE by PBRLM5 were stimulated by the addn. of cytochrome b5. Cytochrome b5 had no effect on CDE prodn. by PBRLM4 and inhibited the prodn. of both CTE and CDE by RLM2. The 2-electron redn. of halothane by cytochrome P 450 was catalyzed by the PBRLM5 form and cytochrome b5 stimulated the transfer of the 2nd electron to halothane through PBRLM5, but not RLM2 or PBRLM4.123Trudell, J. R.; Bosterling; Trevor, A. 1-Chloro-2,2,2-trifluoroethyl radical: Formation from halothane by human cytochrome P-450 in reconstituted vesicles and binding to phospholipids. Biochem. Biophys. Res. Commun. 1981, 102, 372– 377, DOI: 10.1016/0006-291X(81)91531-X[Crossref], [PubMed], [CAS], Google Scholar123https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXlvF2nsbg%253D&md5=41f098c996edd86bf03c48996390dbe81-Chloro-2,2,2-trifluoroethyl radical: formation from halothane by human cytochrome P-450 in reconstituted vesicles and binding to phospholipidsTrudell, James R.; Boesterling, Bernhard; Trevor, AnthonyBiochemical and Biophysical Research Communications (1981), 102 (1), 372-7CODEN: BBRCA9; ISSN:0006-291X.The first complete structural characterization of a metabolically-produced halocarbon radical bound to a phospholipid is reported. Human cytochrome P-450 and NADPH cytochrome P-450 reductase were reconstituted into vesicles composed of dioleoylphosphatidylcholine and egg phosphatidylethanolamine. The vesicles were incubated under Ar with NADPH and [14C]halothane (1-[14C]-2-bromo-2-chloro-1,1,1-trifluoroethane), and the dioleoylphosphatidylcholine fraction was isolated and subjected to transesterification. Sepn. of the resulting fatty acid Me esters resulted in 1 radioactive fraction which gas chromatog.-mass spectrometry revealed to be a mixt. of 9- and 10-(1-chloro-2,2,2-trifluoroethyl)-stearate Me ester.124Pohl, L. R.; Kenna, J. G.; Satoh, H.; Christ, D.; Martin, J. L. Neoantigens associated with halothane hepatitis. Drug Metab. Rev. 1989, 20, 203– 217, DOI: 10.3109/03602538909103537[Crossref], [PubMed], [CAS], Google Scholar124https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXhvQ%253D%253D&md5=eae09f2a981df2b150bcadca6c7de3dfNeoantigens associated with halothane hepatitisPohl, Lance R.; Kenna, J. G.; Satoh, Hiroko; Christ, David; Martin, Jackie L.Drug Metabolism Reviews (1989), 20 (2-4), 203-17CODEN: DMTRAR; ISSN:0360-2532.A review with 33 refs. with discussion on the current status of the nature of the neoantigens assocd. with halothane hepatitis. It is believed that the approaches used to characterize these neoantigens can be applied to the study of the neoantigens assocd. with other drug toxicities believed to have an immunopathol. etiol.125Brown, A. P.; Hastings, K. L.; Gandolfi, A. J.; Brendel, K. Covalent binding of a halothane metabolite and neoantigen production in guinea pig liver slices. Adv. Exp. Med. Biol. 1991, 283, 693– 697, DOI: 10.1007/978-1-4684-5877-0_89[Crossref], [PubMed], [CAS], Google Scholar125https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXlslCnt7c%253D&md5=390bf5d89a3a1ae19487724bb9b33abbCovalent binding of a halothane metabolite and neoantigen production in guinea pig liver slicesBrown, Alan P.; Hastings, Kenneth L.; Gandolfi, A. Jay; Brendel, KlausAdvances in Experimental Medicine and Biology (1991), 283 (Biol. React. Intermed. 4), 693-7CODEN: AEMBAP; ISSN:0065-2598.Halothane hepatitis is a rare and potentially fatal consequence to the use of this anesthetic. The physiol. basis of the disease appears to be an immune response to neoantigens formed by the covalent binding of halothane metabolites to liver protein. Liver slices were used to study the condition for halothane assocd. neoantigen formation in vitro. Liver slices, (1 cm diam., 300 μm thick) from male Harley guinea pigs (600 g) were exposed to either 1.0 or 1.7 mM halothane (media Harley guinea pigs (600 g) were exposed to either 1.0 or 1.7 mM halothane (media concn.) in 95% O2/5% CO2 for 12 h. Covalent binding was detd. using 14C-halothane. Neoantigens were detected by western immunoblot assay using rabbit anti-trifluoroacetylated albumin antiserum. Covalent binding was detected by 1 h of incubation and increased linearly through 12 h. Covalent binding preceded and correlated with the appearance of neoantigen. By 12 h of incubation, 5 neoantigens were seen with mol. wts. ranging from 51-97 kD. These neoantigens have mol. wts. similar to those seen in vivo. This in vitro model system can be used to examine the mechanism for covalent binding and neoantigen prodn. in the hepatocyte.126Brown, A. P.; Hastings, K. L.; Gandolfi, A. J.; Liebler, D. C.; Brendel, K. Formation and identification of protein adducts to cytosolic proteins in guinea pig liver slices exposed to halothane. Toxicology 1992, 73, 281– 295, DOI: 10.1016/0300-483X(92)90070-U[Crossref], [PubMed], [CAS], Google Scholar126https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xlt1Kisrc%253D&md5=3043e5ae3c5da824fcb679d75ead9d04Formation and identification of protein adducts to cytosolic proteins in guinea pig liver slices exposed to halothaneBrown, Alan P.; Hastings, Kenneth L.; Gandolfi, A. Jay; Liebler, Daniel C.; Brendel, KlausToxicology (1992), 73 (3), 281-95CODEN: TXCYAC; ISSN:0300-483X.An in vitro liver slice system was used to study the formation of protein adducts following exposure to halothane. Liver slices (30-35 mg wet wt., 250-300 μm thick) from adult male Hartley guinea pigs (600-800 g) were exposed to [14C]halothane (0.6-0.9 μCi, 1.0-1.7 mM) in 95% O2/5% CO2 for 1, 6 and 12 h. The slices were homogenized and subcellular fractions prepd. Proteins were resolved by electrophoresis and bound radioactivity was detected by scintillation counting and autoradiog. Greater than 80% of detectable radioactivity to whole liver cell protein was localized in the 20-30 kDa range and increased in a linear fashion over the 12-h incubation period. Covalent binding was localized to two proteins of 27 kDa and 26 kDa present in the cytosolic compartment. Purifn. followed by N-terminal amino acid sequence anal. of the 27-kDa protein has identified it to be homologous with glutathione S-transferase. This cytosolic protein appears to be the major target for trifluoroacetylation in liver slices exposed to halothane.127Harris, J. W.; Pohl, L. R.; Martin, J. L.; Anders, M. W. Tissue acylation by the chlorofluorocarbon substitute 2,2-dichloro-1,1,1-trifluoroethane. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 1407– 1410, DOI: 10.1073/pnas.88.4.1407[Crossref], [PubMed], [CAS], Google Scholar127https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXhtFKmu7Y%253D&md5=a0503049d6cb83b8bdca918b54b78988Tissue acylation by the chlorofluorocarbon substitute 2,2-dichloro-1,1,1-trifluoroethaneHarris, James W.; Pohl, Lance R.; Martin, Jackie L.; Anders, M. W.Proceedings of the National Academy of Sciences of the United States of America (1991), 88 (4), 1407-10CODEN: PNASA6; ISSN:0027-8424.Here the authors report studies on the bioactivation of the chlorofluorocarbon substitute 2,2-dichloro-1,1,1-trifluoroethane (HCFC-123) to an electrophilic intermediate that reacts covalently with liver proteins. HCFC-123 and its analog halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) were studied in rats by 19F NMR spectroscopy, and it was found that a trifluoroacetylated lysine adduct was formed with liver proteins. Also, the pattern of proteins immunoreactive with hapten-specific antitrifluoroacetylprotein antibodies was identical in livers of HCFC-123- and halothane-exposed rats. Because halothane causes an idiosyncratic, and sometimes fatal, hepatitis that is assocd. with an immune response against several trifluoroacetylated liver proteins, the present findings raise the possibility that humans exposed to HCFC-123 or structurally related hydrochlorofluorocarbons may be at risk of developing an immunol. mediated hepatitis.128Pohl, L. R. An immunochemical approach of identifying and characterizing protein targets of toxic reactive metabolites. Chem. Res. Toxicol. 1993, 6, 786– 793, DOI: 10.1021/tx00036a006[ACS Full Text], [CAS], Google Scholar128https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXmsVejsLs%253D&md5=30dd61034178e27597f0202a2dee0f0eAn immunochemical approach of identifying and characterizing protein targets of toxic reactive metabolitesPohl, Lance R.Chemical Research in Toxicology (1993), 6 (6), 786-93CODEN: CRTOEC; ISSN:0893-228X.A review with 101 refs. on protein adducts assocd. with halothane hepatitis, other uses of anti-TFA antibodies, protein adducts assocd. with acetaminophen hepatotoxicity, protein adducts assocd. with ethanol hepatotoxicity, protein adducts assocd. with diclofenac hepatotoxicity, and proteins posttranslationally modified by products of oxidative stress.129Kenna, J. G.; Satoh, H.; Christ, D. D.; Pohl, L. R. Metabolic basis for a drug hypersensitivity: antibodies in sera from patients with halothane hepatitis recognize liver neoantigens that contain the trifluoroacetyl group derived from halothane. J. Pharmacol. Exp. Ther. 1988, 245, 1103– 1109[PubMed], [CAS], Google Scholar129https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXks1KktbY%253D&md5=23dad774921d26a4aaa46b06e7a1a748Metabolic basis for a drug hypersensitivity: antibodies in sera from patients with halothane hepatitis recognize liver neoantigens that contain the trifluoroacetyl group derived from halothaneKenna, J. G.; Satoh, H.; Christ, D. D.; Pohl, L. R.Journal of Pharmacology and Experimental Therapeutics (1988), 245 (3), 1103-9CODEN: JPETAB; ISSN:0022-3565.Liver microsomes form rats treated in vivo with halothane or deuterated halothane were tested by immunoblotting for reactivity with patients' sera and with an antiserum specific for the covalently bound trifluoroacetyl (TFA) halide metabolite of halothane. Rat liver microsomes incubated aerobically or anaerobically with halothane or deuterated halothane in vitro, ± NADPH and/or NADH, were also analyzed. Neoantigen expression involved oxidative halothane metab. by cytochromes P 450 to TFA halide and covalent binding of the TFA group to the proteins. Incubation of microsomes from halothane-treated rats with 1 M piperidine cleaved the TFA groups from the proteins and abolished antigenicity, confirming this conclusion. Recognition of the neoantigens by the patients' antibodies was inhibited only partially by the hapten deriv. N-E-TFA-Dl-lysine. It appears that the patients' antibodies recognize epitopes consisting of the TFA group plus assocd. structural features of the protein carriers (100 kilodalton (kDa), 76 kDa, 59 kDa, 57 kDa, and 54 kDa), not the TFA hapten alone. The approach described may be of general utility for characterization of drug-induced neoantigens assocd. with other drug hypersensitivities.130Kenna, J. G.; Neuberger, J.; Williams, R. Evidence for expression in human liver of halothane-induced neoantigens recognized by antibodies in sera from patients with halothane hepatitis. Hepatology 1988, 8, 1635– 1641, DOI: 10.1002/hep.1840080627[Crossref], [PubMed], [CAS], Google Scholar130https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaL1M%252FltlansA%253D%253D&md5=d41d0e21185e3bcd138268cb3fb20805Evidence for expression in human liver of halothane-induced neoantigens recognized by antibodies in sera from patients with halothane hepatitisKenna J G; Neuberger J; Williams RHepatology (Baltimore, Md.) (1988), 8 (6), 1635-41 ISSN:0270-9139.Previous investigations have shown that antibodies in sera from patients with halothane hepatitis recognize neoantigens, expressed in livers of halothane-exposed rabbits and rats, which consist of a halothane metabolite bound covalently to specific microsomal proteins. These studies have suggested that the patients' antibodies may play a role in the pathogenesis of the hepatitis. In the present investigation, human liver biopsy samples were analyzed using an immunoblotting method to seek evidence for expression of halothane-induced neoantigens in humans. Sera from four patients with halothane hepatitis, which recognized halothane-induced rabbit liver neoantigens of 100, 76 and 57 kD, reacted strongly with antigens of very similar molecular weights that were expressed in livers from two patients who had died of cardiac failure following recent anesthesia with halothane. The antigens were not expressed in normal human liver or in livers from three patients who died of cardiac failure following anesthesia with agents other than halothane. The human antigens were not recognized by antibodies present in various control sera. Recognition of the 100- and 76-kD human antigens by the patients' antibodies was greatly reduced by absorption of sera with liver microsomes from halothane-exposed rabbits, but not by absorption of sera with control rabbit microsomes. These results indicate that humans exposed to halothane express liver neoantigens which are analogous to the halothane metabolite-protein neoantigens characterized previously in halothane-exposed animals.131Behne, M.; Wilke, H. J.; Harder, S. Clinical pharmacokinetics of sevoflurane. Clin. Pharmacokinet. 1999, 36, 13– 26, DOI: 10.2165/00003088-199936010-00002[Crossref], [PubMed], [CAS], Google Scholar131https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXht1WgsbY%253D&md5=038daca584abfd368886530bd6a83d53Clinical pharmacokinetics of sevofluraneBehne, Michael; Wilke, Hans-Joachim; Harder, SebastianClinical Pharmacokinetics (1999), 36 (1), 13-26CODEN: CPKNDH; ISSN:0312-5963. (Adis International Ltd.)Sevoflurane is a comparatively recent addn. to the range of inhalational anesthetics which has been recently released for clin. use. In comparison to older inhalational agents such as isoflurane or halothane, the most important property of sevoflurane is its low soly. in the blood. This results in a more rapid uptake and induction than the "older" inhalational agents, improved control of depth of anesthesia and faster elimination and recovery. The more rapid pharmacokinetics are a result of the low blood/gas partition coeff. of 0.69. With an oil/gas partition coeff. of 47.2, the min. alveolar concn. (MAC) of sevoflurane is 2.05%. Two to 5% of the drug taken up is metabolized by the liver. The pharmacokinetics of sevoflurane do not change in children, obese patients or patients with renal insufficiency. The pharmacokinetics and pleasant odor of sevoflurane make mask induction feasible, which is an obvious advantage in pediatric anesthesia. The hepatic metab. of sevoflurane results in the formation of inorg. fluoride. Upon contact with alk. CO2 absorbent, a small amt. of sevoflurane is degraded and a metabolite (compd. A) is formed and inhaled in trace amts. Whether inorg. fluoride or compd. A are nephrotoxic is presently a matter of controversy.132Kharasch, E. D.; Karol, M. D.; Lanni, C.; Sawchuk, R. Clinical sevoflurane metabolism and disposition. I. Sevoflurane and metabolite pharmacokinetics. Anesthesiology 1995, 82, 1369– 1378, DOI: 10.1097/00000542-199506000-00008[Crossref], [PubMed], [CAS], Google Scholar132https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK2Mzhtl2lug%253D%253D&md5=1b52ac6489fe4d61e555aa26de0cfc6eClinical sevoflurane metabolism and disposition. I. Sevoflurane and metabolite pharmacokineticsKharasch E D; Karol M D; Lanni C; Sawchuk RAnesthesiology (1995), 82 (6), 1369-78 ISSN:0003-3022.BACKGROUND: Sevoflurane has low blood and tissue solubility and is metabolized to free fluoride and hexafluoroisopropanol (HFIP). Although sevoflurane uptake and distribution and fluoride formation have been described, the pharmacokinetics of HFIP formation and elimination are incompletely understood. This investigation comprehensively characterized the simultaneous disposition of sevoflurane, fluoride, and HFIP. METHODS: Ten patients within 30% of ideal body weight who provided institutional review board-approved informed consent received sevoflurane (2.7% end-tidal, 1.3 MAC) in oxygen for 3 h after propofol induction, after which anesthesia was maintained with propofol, fentanyl, and nitrous oxide. Sevoflurane and unconjugated and total HFIP concentrations in blood were determined during anesthesia and for 8 h thereafter. Plasma and urine fluoride and total HFIP concentrations were measured during and through 96 h after anesthetic administration. Fluoride and HFIP were quantitated using an ion-selective electrode and by gas chromatography, respectively. RESULTS: The total sevoflurane dose, calculated from the pulmonary uptake rate, was 88.8 +/- 9.1 mmol. Sevoflurane was rapidly metabolized to the primary metabolites fluoride and HFIP, which were eliminated in urine. HFIP circulated in blood primarily as a glucuronide conjugate, with unconjugated HFIP < or = 15% of total HFIP concentrations. In blood, peak unconjugated HFIP concentrations were less than 1% of peak sevoflurane concentrations. Apparent renal fluoride and HFIP clearances (mean +/- SE) were 51.8 +/- 4.5 and 52.6 +/- 6.1 ml/min, and apparent elimination half-lives were 21.4 +/- 2.8 and 20.1 +/- 2.6 h, respectively. Renal HFIP and net fluoride excretion were 4,300 +/- 540 and 3,300 +/- 540 mumol. Compared with the estimated sevoflurane uptake, 4.9 +/- 0.5% of the dose taken up was eliminated in the urine as HFIP. For fluoride, 3.7 +/- 0.4% of the sevoflurane dose taken up was eliminated in the urine, which, because a portion of fluoride is sequestered in bone, corresponded to approximately 5.6% of the sevoflurane dose metabolized to fluoride. CONCLUSIONS: Sevoflurane was rapidly metabolized to fluoride and HFIP, which was rapidly glucuronidated and eliminated in the urine. The overall extent of sevoflurane metabolism was approximately 5%.133Kharasch, E. D.; Armstrong, A. S.; Gunn, K.; Artru, A.; Cox, K.; Karol, M. D. Clinical sevoflurane metabolism and disposition. II. The role of cytochrome P450 2E1 in fluoride and hexafluoroisopropanol formation. Anesthesiology 1995, 82, 1379– 1388, DOI: 10.1097/00000542-199506000-00009[Crossref], [PubMed], [CAS], Google Scholar133https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK2Mzhtl2luw%253D%253D&md5=373c22f4ccb4ff24b6df206307dc67d1Clinical sevoflurane metabolism and disposition. II. The role of cytochrome P450 2E1 in fluoride and hexafluoroisopropanol formationKharasch E D; Armstrong A S; Gunn K; Artru A; Cox K; Karol M DAnesthesiology (1995), 82 (6), 1379-88 ISSN:0003-3022.BACKGROUND: Sevoflurane is metabolized to free fluoride and hexafluoroisopropanol (HFIP). Cytochrome P450 2E1 is the major isoform responsible for sevoflurane metabolism by human liver microsomes in vitro. This investigation tested the hypothesis that P450 2E1 is predominantly responsible for sevoflurane metabolism in vivo. Disulfiram, which is converted in vivo to a selective inhibitor of P450 2E1, was used as a metabolic probe for P450 2E1. METHODS: Twenty-one patients within 30% of ideal body weight, who provided institutional review board-approved informed consent and were randomized to receive disulfiram (500 mg oral, n = 11) or nothing (control, n = 10) the night before surgery, were evaluated. All patients received sevoflurane (2.7% end-tidal, 1.3 MAC) in oxygen for 3 h after propofol induction. Thereafter, sevoflurane was discontinued, and anesthesia was maintained with propofol, fentanyl, and nitrous oxide. Blood sevoflurane concentrations during anesthesia and for 8 h thereafter were measured by gas chromatography. Plasma and urine fluoride and total (unconjugated plus glucuronidated) HFIP concentrations were measured by an ion-selective electrode and by gas chromatography, respectively, during anesthesia and for 96 h postoperatively. RESULTS: Patient groups were similar with respect to age, weight, sex, case duration, and intraoperative blood loss. The total sevoflurane dose, measured by cumulative end-tidal sevoflurane concentrations (3.7 +/- 0.1 MAC-h; mean +/- SE), total pulmonary uptake, and blood sevoflurane concentrations, was similar in both groups. In control patients, plasma fluoride and HFIP concentrations were increased compared to baseline values intraoperatively and postoperatively for the first 48 and 60 h, respectively. Disulfiram treatment significantly diminished this increase. Plasma fluoride concentrations increased from 2.1 +/- 0.3 microM (baseline) to 36.2 +/- 3.9 microM (peak) in control patients, but only from 1.7 +/- 0.2 to 17.0 +/- 1.6 microM in disulfiram-treated patients (P < 0.05 compared with control patients). Peak plasma HFIP concentrations were 39.8 +/- 2.6 and 14.4 +/- 1.1 microM in control and disulfiram-treated patients (P < 0.05), respectively. Areas under the plasma fluoride- and HFIP-time curves also were diminished significantly to 22% and 20% of control patients, respectively, by disulfiram treatment. Urinary excretion of fluoride and HFIP was similarly significantly diminished in disulfiram-treated patients. Cumulative 96-h fluoride and HFIP excretion in disulfiram-treated patient was 1,080 +/- 210 and 960 +/- 240 mumol, respectively, compared to 3,950 +/- 560 and 4,300 +/- 540 mumol in control patients (P < 0.05). CONCLUSIONS: Disulfiram, an effective P450 2E1 inhibitor, substantially decreased fluoride ion and HFIP production during and after sevoflurane anesthesia. These results suggest that P450 2E1 is a predominant P450 isoform responsible for human sevoflurane metabolism in vivo.134Kharasch, E. D. Biotransformation of sevoflurane. Anesth. Analg. 1995, 81, 27S– 38S, DOI: 10.1097/00000539-199512001-00005[Crossref], [PubMed], [CAS], Google Scholar134https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XmvV2rtQ%253D%253D&md5=5ebb6765156a1c6cc2398213af8a4bfbBiotransformation of sevofluraneKharasch, Evan D.Anesthesia & Analgesia (Baltimore) (1995), 81 (6S), S27-S38CODEN: AACRAT; ISSN:0003-2999. (Williams & Wilkins)A review with 68 refs. Biotransformation of volatile anesthetics is of considerable interest because of known assocns. between metab. of certain anesthetics and specific organ toxicities. Advances in enzyme biochem. and mol. biol. have illuminated similarities and differences between animal and human drug metabolizing enzymes, permitting both appropriate extrapolation of animal data to humans and reconciliation of discrepancies between animal and human sevoflurane studies.135Thummel, K. E.; Kharasch, E. D.; Podoll, T.; Kunze, K. Human liver microsomal enflurane defluorination catalyzed by cytochrome P-450 2E1. Drug Metab. Dispos. 1993, 21, 350– 357[PubMed], [CAS], Google Scholar135https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXisVKrurY%253D&md5=1e44133d539b2b04e71d6845f0f438aeHuman liver microsomal enflurane defluorination catalyzed by cytochrome P-450 2E1Thummel, Kenneth E.; Kharasch, Evan D.; Podoll, Terry; Kunze, KentDrug Metabolism and Disposition (1993), 21 (2), 350-7CODEN: DMDSAI; ISSN:0090-9556.The volatile anesthetic agent enflurane undergoes oxidative metab. in human liver, yielding both inorg. and org. fluoride metabolites. Numerous studies in animals indicate that cytochrome P 450 2E1 is a major catalyst for the defluorination reaction. However, the P 450 enzyme catalyzing enflurane metab. in humans has not been identified. Therefore, expts. were conducted to det. whether hepatic P 450 2E1 is a catalyst for the reaction in humans, and whether other constitutive or inducible isoforms might also be involved. Purified human liver P 450 2E1, reconstituted with cytochrome b5 and P 450 reductase, catalyzed enflurane defluorination at a rate of 9.3 nmol F-/nmol P 450/30 min, in contrast to a mean liver microsomal rate of 2.0 nmol F-/nmol P 450/30 min. The microsomal rate of defluorination for individual human livers correlated significantly with the microsomal content of P 450 2E1 protein, the rate of p-nitrophenol hydroxylation, and the rate of chlorzoxazone 6-hydroxylation. In addn., specific anti-P 450 2E1 IgG, at a concn. of 10 mg IgG/nmol P 450, inhibited the microsomal reaction by 80%. Finally, a series of P 450 isoform-specific chem. inhibitors of oxidative metab. were screened for their ability to block human microsomal enflurane defluorination. Only di-Et dithiocarbamate, a mechanism-based inhibitor of P 450 2E1, inhibited the reaction; this occurred to a degree comparable to the effect of anti-P 450 2E1 antibody. These results demonstrate that P 450 2E1 is the predominant, if not only, enzyme catalyzing enflurane defluorination in human liver.136Kharasch, E. D.; Thummel, K. E.; Mautz, D.; Bosse, S. Clinical enflurane metabolism by cytochrome P450 2E1. Clin. Pharmacol. Ther. 1994, 55, 434– 440, DOI: 10.1038/clpt.1994.53[Crossref], [PubMed], [CAS], Google Scholar136https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK2c3hsFarsw%253D%253D&md5=75c256e631e235ca79b283322d545e32Clinical enflurane metabolism by cytochrome P450 2E1Kharasch E D; Thummel K E; Mautz D; Bosse SClinical pharmacology and therapeutics (1994), 55 (4), 434-40 ISSN:0009-9236.BACKGROUND: Fluorinated ether anesthetic hepatotoxicity and nephrotoxicity are mediated by cytochrome P450-catalyzed oxidative metabolism. Metabolism of the volatile anesthetic enflurane to inorganic fluoride ion by human liver microsomes in vitro is catalyzed predominantly by the cytochrome P450 isoform CYP2E1. This investigation tested the hypothesis that P450 2E1 is also the isoform responsible for human enflurane metabolism in vivo. Disulfiram, which is converted in vivo to a selective inhibitor of P450 2E1, was used as a metabolic probe for P450 2E1. METHODS: Twenty patients undergoing elective surgery were randomized to receive disulfiram (500 mg orally; n = 10) or nothing (control subjects; n = 10) the evening before surgery. All patients received a standard anesthetic of enflurane (2.2% end-tidal) in oxygen for 3 hours. Blood enflurane concentrations were measured by gas chromatography. Plasma and urine fluoride concentrations were quantitated by ion-selective electrode. RESULTS: Patient groups were similar with respect to age, weight, gender, duration of surgery, and blood loss. Total enflurane dose, measured by cumulative end-tidal enflurane concentrations (3.9 to 4.1 MAC-hr) and by blood enflurane concentrations, was similar in both groups. Plasma fluoride concentrations increased from 3.6 +/- 1.5 mumol/L (baseline) to 24.3 +/- 3.8 mumol/L (peak) in untreated patients (mean +/- SE). Disulfiram treatment completely abolished the rise in plasma fluoride concentration. Urine fluoride excretion was similarly significantly diminished in disulfiram-treated patients. Fluoride excretion in disulfiram-treated patients was 62 +/- 10 and 61 +/- 12 mumol on days 1 and 2, respectively, compared with 1090 +/- 180 and 1200 +/- 220 mumol in control subjects (p < 0.05 on each day). CONCLUSIONS: Disulfiram prevented fluoride ion production after enflurane anesthesia. These results suggest that P450 2E1 is the predominant P450 isoform responsible for human clinical enflurane metabolism in vivo.137Kharasch, E. D.; Hankins, D. C.; Cox, K. Clinical isoflurane metabolism by cytochrome P450 2E1. Anesthesiology 1999, 90, 766– 771, DOI: 10.1097/00000542-199903000-00019[Crossref], [PubMed], [CAS], Google Scholar137https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXhvFKhur4%253D&md5=4b4c448ff90ee438102a0aafdf520d67Clinical isoflurane metabolism by cytochrome P450 2E1Kharasch, Evan D.; Hankins, Douglas C.; Cox, KathyAnesthesiology (1999), 90 (3), 766-771CODEN: ANESAV; ISSN:0003-3022. (Lippincott Williams & Wilkins)Some evidence suggests that isoflurane metab. to trifluoroacetic acid and inorg. fluoride by human liver microsomes in vitro is catalyzed by cytochrome P 450 2E1 (CYP2E1). This investigation tested the hypothesis that P 450 2E1 predominantly catalyzes human isoflurane metab. in vivo. Disulfiram, which is converted in vivo to a selective inhibitor of P 450 2E1, was used as a metabolic probe for P 450 2E1. Twenty-two elective surgery patients who provided institutionally-approved written informed consent were randomized to receive disulfiram (500 mg orally, N = 12) or nothing (controls, N = 10) the evening before surgery. All patients received a std. isoflurane anesthetic (1.5% end-tidal in oxygen) for 8 h. Urine and plasma trifluoroacetic acid and fluoride concns. were quantitated in samples obtained for 4 days postoperatively. Patient groups were similar with respect to age, wt., gender, duration of surgery, blood loss, and delivered isoflurane dose, measured by cumulative end-tidal isoflurane concns. (9.7-10.2 MAC-hr). Postoperative urine excretion of trifluoroacetic acid (days 1-4) and fluoride (days 1-3) was significantly (P < 0.05) diminished in disulfiram-treated patients. Cumulative 0-96 h excretion of trifluoroacetic acid and fluoride in disulfiram-treated patients was 34 ± 72 and 270 ± 70 μmoles (mean ± SD), resp., compared to 440 ± 360 and 1500 ± 800 μmoles in controls (P < 0.05 for both). Disulfiram also abolished the rise in plasma metabolite concns. Disulfiram, a selective inhibitor of human hepatic P 450 2E1, prevented 80-90% of isoflurane metab. These results suggest that P 450 2E1 is the predominant P 450 isoform responsible for human clin. isoflurane metab. in vivo.138Strum, D. P.; Johnson, B. H.; Eger, E. I. 2nd. Stability of sevoflurane in soda lime. Anesthesiology 1987, 67, 779– 781, DOI: 10.1097/00000542-198711000-00024[Crossref], [PubMed], [CAS], Google Scholar138https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXhtFWisg%253D%253D&md5=8d8faef9112c85c70c188ee729f719d0Stability of sevoflurane in soda limeStrum, David P.; Johnson, Brynte H.; Eger, Edmond I., IIAnesthesiology (1987), 67 (5), 779-81CODEN: ANESAV; ISSN:0003-3022.Stability of a halogenated volatile anesthetic is important because of the potential toxicity assocd. with the breakdown products. A temp.-dependent degrdn. of sevoflurane [(F3C)2CHOCH2F] in soda lime was obsd. At 22°, soda lime degraded 6.5% of the sevoflurane per h. The rate increased by 1.6% per h per degree rise in temp., reaching 57.4% degrdn. per h at 54°. In contrast, isoflurane was not degraded by soda lime. Halothane did not degrade at 22° or 37°, but did degrade (2.2% per h) at 54°.139Morio, M.; Fujii, K.; Satoh, N.; Imai, M.; Kawakami, U.; Mizuno, T.; Kawai, Y.; Ogasawara, Y.; Tamura, T.; Negishi, A.; Kumagai, Y.; Kawai, Y. Reaction of sevoflurane and its degradation products with soda lime. Toxicity of the byproducts. Anesthesiology 1992, 77, 1155– 1164, DOI: 10.1097/00000542-199212000-00017[Crossref], [PubMed], [CAS], Google Scholar139https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXpsVWrug%253D%253D&md5=7108d6c9e328915a816593c6c5f14740Reaction of sevoflurane and its degradation products with soda lime. Toxicity of the byproductsMorio, Michio; Fujii, Kohyu; Satoh, Nobukatsu; Imai, Masahiro; Kawakami, Urao; Mizuno, Takahiro; Kawai, Yoichiro; Ogasawara, Yasumasa; Tamura, Takashi; et al.Anesthesiology (1992), 77 (6), 1155-64CODEN: ANESAV; ISSN:0003-3022.Sevoflurane previously has been reported to undergo extensive degrdn. in the presence of soda lime. To more completely characterize the extent and significance of this reaction, the authors studied degrdn. of sevoflurane with and without soda lime, as well as the toxicity and mutagenicity of the degrdn. products. Two degrdn. products detected were CF2 = C(CF3)OCH2F (compd. A) and CH3OCF2CH(CF3)OCH2F (compd. B). During circulation of 1%, 2%, and 3% sevoflurane in a closed anesthesia circuit for 8 h, peak concns. of compd. A were 13.3, 30.2, and 42.1 ppm at 2 h, resp. The concns. of compd. B did not exceed 2 ppm. The temp. of the soda lime was 43.3° at 1 h and increased gradually to 47.9° after 8 h. In closed flasks with soda lime, the magnitude of the decrease in sevoflurane concns. (3%) and of the increase in compd. A concns. was temp. dependent. The peak concns. of compd. A at 23°, 37°, and 54° were 32.8 at 2 h, 46.6 at 0.5 h, and 78.5 ppm at 0.5 h, resp. The LC50 (50% lethal concn.) of compd. A in Wistar rats was 1090 ppm in males and 1050 ppm in females exposed for 1 h. The LC50 was 420 ppm in males and 400 ppm in females exposed for 3 h. The chronic toxicity of compd. A in Wistar rats was studied by exposing rats 24 times, for 3 h each, to initial concns. of 30, 60, or 120 ppm in a ventilated chamber. At all concns., there were no apparent effects other than a loss of body wt. in females (120 ppm) on the final day. Compd. A did not induce mutation on the reverse (Ames) test at <2500 μg/dish (culture medium 2.7 mL) with activation by S-9 mixt., and below 1250 μg/dish (culture medium 2.7 mL) without activation, in four strains of S. typhimurium and in 1 strain of E. coli. Exposure of fibroblasts to 7500 ppm of compd. A for 1 h did not induce structural changes. In a study of acute toxicity of compd. B, there was no toxicity in Wistar rats after 3 h of exposure at 2400 ppm. The reverse (Ames) test for compd. B was neg. at 625-1250 μg/dish. The authors conclude that sevoflurane is extensively degraded in the presence of soda lime and heat and the LC50 in rats of one degrdn. product is >10-fold the peak concn. reached during an 8-h closed circuit anesthetic.140Jin, L.; Davis, M. R.; Kharasch, E. D.; Doss, G. A.; Baillie, T. A. Identification in rat bile of glutathione conjugates of fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether, a nephrotoxic degradate of the anesthetic agent sevoflurane. Chem. Res. Toxicol. 1996, 9, 555– 561, DOI: 10.1021/tx950162m[ACS Full Text], [CAS], Google Scholar140https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xpt1agsg%253D%253D&md5=c5c91c6633610cc7a80c031ee5726086Identification in rat bile of glutathione conjugates of fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether, a nephrotoxic degradate of the anesthetic agent sevofluraneJin, Lixia; Davis, Margaret R.; Kharasch, Evan D.; Doss, George A.; Baillie, Thomas A.Chemical Research in Toxicology (1996), 9 (2), 555-61CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)Recent studies have indicated that the nephrotoxicity of fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether ("Compd. A"), a breakdown product of the inhaled anesthetic sevoflurane, may be mediated by a reactive intermediate(s) generated via the cysteine conjugate β-lyase pathway. To gain a better understanding of glutathione (GSH)-dependent metab. of Compd. A, the present study was carried out with the primary goal of detecting and characterizing Compd. A-GSH conjugates. By ionspray LC-MS/MS and NMR spectroscopy, a total of four GSH conjugates ("A1-A4") were identified from the bile of rats dosed i.p. with Compd. A. A1 and A2 were identified as two diastereomers of S-[1,1-difluoro-2-(fluoromethoxy)-2-(trifluoromethyl)ethyl]glutathione, while A3 and A4 were identified as (E)- and (Z)-S-[1-fluoro-2-(fluoromethoxy)-2-(trifluoromethyl)vinyl]glutathione, resp. Quant. analyses indicated that approx. 29% of the administered dose of Compd. A was excreted into the bile in the form of the above GSH conjugates over a period of 6 h. Studies conducted in vitro demonstrated that the reaction of Compd. A with GSH was catalyzed by both rat liver cytosolic and microsomal glutathione S-transferases (GST), with the two enzyme systems exhibiting different product selectivities. Formation of these GSH conjugates also occurred nonenzymically at an appreciable rate. These results indicate that spontaneous and enzyme-mediated conjugation with GSH represents a major pathway of metab. of Compd. A in rats. Conjugation of Compd. A with GSH in vivo appeared to be catalyzed preferentially by microsomal rather than cytosolic GST, based on comparison of biliary, microsomal, and cytosolic metabolic profiles. By analogy with other haloalkenes, further metab. of the corresponding cysteine conjugates of Compd. A by renal cysteine conjugate β-lyase may lead to the formation of reactive acylating agents, which would be expected to bind covalently to cellular macromols. and cause organ-selective nephrotoxicity.141Iyer, R. A.; Anders, M. W. Cysteine conjugate β-lyase-dependent biotransformation of the cysteine S-conjugates of the sevoflurane degradation product 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A). Chem. Res. Toxicol. 1997, 10, 811– 819, DOI: 10.1021/tx960196+[ACS Full Text], [CAS], Google Scholar141https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXktVSisrk%253D&md5=df9198d23d3512e8831bceb07db0b391Cysteine Conjugate β-Lyase-Dependent Biotransformation of the Cysteine S-Conjugates of the Sevoflurane Degradation Product 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (Compound A)Iyer, Ramaswamy A.; Anders, M. W.Chemical Research in Toxicology (1997), 10 (7), 811-819CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (I, Compd. A) is a fluoroalkene formed by the base-catalyzed degrdn. of sevoflurane and is nephrotoxic in rats. Fluoroalkene I is a structural analog of other nephrotoxic haloalkenes that undergo glutathione S-conjugate formation and cysteine S-conjugate β-lyase-dependent bioactivation to reactive intermediates. The present expts. were designed to study the β-lyase-dependent biotransformation of S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine (II) and S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine by 19F NMR and UV spectroscopy and GC/MS. Incubation of cysteine S-conjugate II with rat kidney cytosol or a pyridoxal model system showed the formation of inorg. fluoride, pyruvate, and 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid (III), the expected products of a β-lyase-catalyzed reaction. The ratio of fluoride to pyruvate ranged from 2.3 to 2.5. The amt. of acid III formed in the rat kidney cytosol and the pyridoxal model system was, however, less than 5% of the amt. of pyruvate formed. Incubation of conjugate II with rat kidney cytosol and anal. by 19F NMR spectroscopy showed resonances that were assigned to 3,3,3-trifluorolactic acid (IV); the formation of acid IV was obsd. in the pyridoxal model only after prolonged incubation (>18 h). Lactic acid IV was identified as a degrdn. product of acid 9. Cysteine S-conjugate III was not stable in pH 7.4 buffer and underwent a rapid cyclization reaction (t1/2 ≈ III min) to form 2-[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3-thiazole-4-carboxylic acid. These data show that fluoroalkene I-derived cysteine S-conjugates are substrates for renal β-lyase and that acid III is formed as a terminal product. Acid III is, however, unstable and affords lactic acid IV as a degrdn. product.142Iyer, R. A.; Frink, E. J., Jr.; Ebert, T. J.; Anders, M. W. Cysteine conjugate β-lyase-dependent metabolism of compound A (2-[fluoromethoxy]-1,1,3,3,3-pentafluoro-1-propene) in human subjects anesthetized with sevoflurane and in rats given compound A. Anesthesiology 1998, 88, 611– 618, DOI: 10.1097/00000542-199803000-00009[Crossref], [PubMed], [CAS], Google Scholar142https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXitFamtrs%253D&md5=386ee386aeeb5d0a53dcc54ade4727dfCysteine conjugate β-lyase-dependent metabolism of compound a (2-[fluoromethoxy]-1,1,3,3,3-pentafluoro-1-propene) in human subjects anesthetized with sevoflurane and in rats given compound AIyer, Ramaswamy A.; Frink, Edward J., Jr.; Ebert, Thomas J.; Anders, M. W.Anesthesiology (1998), 88 (3), 611-618CODEN: ANESAV; ISSN:0003-3022. (Lippincott-Raven Publishers)Sevoflurane undergoes Baralyme- or soda lime-catalyzed degrdn. in the anesthesia circuit to yield compd. A (2-[fluoromethoxy]-1,1,3,3,3-pentafluoro-1-propene), which is nephrotoxic in rats and undergoes metab. via the cysteine conjugate β-lyase pathway in those animals. The objective of these expts. was to test the hypothesis that compd. A undergoes β-lyase-dependent metab. in humans. Human volunteers were anesthetized with sevoflurane (1.25 min. alveolar concn., 3%, 2 l/min, 8 h) and thereby exposed to compd. A. Urine was collected at 24-h intervals for 72 h after anesthesia. Rats, which served as a pos. control, were given compd. A i.p., and urine was collected for 24 h afterward. Human and rat urine samples were analyzed by 19F NMR spectroscopy and gas chromatog.-mass spectrometry for the presence of compd. A metabolites. Anal. of human and rat urine showed the presence of the compd. A metabolites S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-NK-acetyl-L-cysteine, (E)- and (Z)-S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid, 3,3,3-trifluorolactic acid, and inorg. fluoride. The presence of 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid and 3,3,3-trifluorolactic acid in human urine was confirmed by gas chromatog.-mass spectrometry. The formation of compd. A-derived mercapturates shows that compd. A undergoes glutathione S-conjugate formation. The identification of 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid and 3,3,3-trifluorolactic acid in the urine of humans anesthetized with sevoflurane shows that compd. A undergoes β-lyase-dependent metab. Metabolite formation was qual. similar in both human volunteers anesthetized with sevoflurane, and thereby exposed to compd. A, and in rats given compd. A, indicating that compd. A is metabolized by the β-lyase pathway in both species.143Martin, J. L.; Laster, M. J.; Kandel, L.; Kerschmann, R. L.; Reed, G. F.; Eger, E. I., II. Metabolism of compound A by renal cysteine-S-conjugate β-lyase is not the mechanism of compound A-induced renal injury in the rat. Anesth. Analg. 1996, 82, 770– 774, DOI: 10.1213/00000539-199604000-00017[Crossref], [PubMed], [CAS], Google Scholar143https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xis1Wltrw%253D&md5=614ccdeec985860132be925d7d197fcbMetabolism of compound A by renal cysteine-S-conjugate β-lyase is not the mechanism of compound A-induced renal injury in the ratMartin, J. L.; Laster, M. J.; Kandel, L.; Kerschmann, R. L.; Reed, G. F.; Eger, E. I. IIAnesthesia & Analgesia (Baltimore) (1996), 82 (4), 770-4CODEN: AACRAT; ISSN:0003-2999. (Williams & Wilkins)Compd. A [CF2:C(CF3)OCH2F], a vinyl ether produced by CO2 absorbents acting on sevoflurane, can produce corticomedullary junction necrosis (injury to the outer stripe of the outer medullary layer, i.e., corticomedullary junction) in rats. Several halogenated alkenes produce a histol. similar corticomedullary necrosis by converting glutathione conjugates of these alkenes to halothionoacetyl halides. To test whether this mechanism explained the nephrotoxicity of Compd. A, we blocked three metabolic steps which would lead to formation of a halothionoacetyl halide: (1) we depleted glutathione by administering dl-buthionine-S,R-sulfoximine (BSO); (2) we blocked cysteine S-conjugate formation by administering acivicin (AT-125); and (3) we inhibited subsequent metab. by renal cysteine conjugate β-lyase to the nephrotoxic halothionoacetyl halides by administering aminooxyacetic acid (AOAA). These treatments were given alone or in combination to sep. groups of 10 or 20 Wistar rats before their exposure to Compd. A. We hypothesized that blocking these metabolic steps should decrease the injury produced by breathing 150 ppm of Compd. A for 3 h. However, we found either no change or an increase in renal injury, suggesting that this pathway mediates detoxification rather than toxicity. Our findings suggest that the cysteine-S-conjugate-mediated pathway is not the mechanism of Compd. A nephrotoxicity and, therefore, obsd. interspecies differences in the activity of this activating pathway may not be relevant in the prediction of the nephrotoxic potential of Compd. A in clin. practice.144Kharasch, E. D.; Hoffman, G. M.; Thorning, D.; Hankins, D. C.; Kilty, C. G. Role of the renal cysteine conjugate β-lyase pathway in inhaled compound A nephrotoxicity in rats. Anesthesiology 1998, 88, 1624– 1633, DOI: 10.1097/00000542-199806000-00027[Crossref], [PubMed], [CAS], Google Scholar144https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXkt1egsr8%253D&md5=cdd5d6e73c285ad91b3a06fd6d7cd21eRole of the renal cysteine conjugate β-lyase pathway in inhaled compound A nephrotoxicity in ratsKharasch, Evan D.; Hoffman, Gary M.; Thorning, David; Hankins, Douglas C.; Kilty, Cormac G.Anesthesiology (1998), 88 (6), 1624-1633CODEN: ANESAV; ISSN:0003-3022. (Lippincott-Raven Publishers)The sevoflurane degrdn. product compd. A is nephrotoxic in rats and undergoes metab. to glutathione and cysteine S-conjugates, with further metab. by renal cysteine conjugate β-lyase to reactive intermediates. Evidence suggests that toxicity is mediated by renal uptake of compd. A S-conjugates and metab. by β-lyase. Previously, inhibitors of the β-lyase pathway (aminooxyacetic acid and probenecid) diminished the nephrotoxicity of i.p. compd. A. This investigation detd. inhibitor effects on the toxicity of inhaled compd. A. Fischer 344 rats underwent 3 h of nose-only exposure to compd. A (0-220 ppm in initial dose-response expts. and 100-109 ppm in subsequent inhibitor expts.). The inhibitors (and targets) were probenecid (renal org. anion transport mediating S-conjugate uptake), acivicin (γ-glutamyl transferase), aminooxyacetic acid (renal β-lyase), and aminobenzotriazole (cytochrome P 450). Urine was collected for 24 h, and the animals were killed. Nephrotoxicity was assessed by histol. and biochem. markers (serum BUN and creatinine; urine vol.; and excretion of protein, glucose, and α-glutathione-S-transferase, a predominantly proximal tubular cell protein). Compd. A caused dose-related proximal tubular cell necrosis, diuresis, proteinuria, glucosuria, and increased α-glutathione-S-transferase excretion. The threshold for toxicity was 98-109 ppm (294-327 ppm-h). Probenecid diminished compd. A-induced glucosuria and excretion of α-glutathione-S-transferase and completely prevented necrosis. Aminooxyacetic acid diminished compd. A-dependent proteinuria and glucosuria but did not decrease necrosis. Acivicin increased nephrotoxicity of compd. A, and aminobenzotriazole had no consistent effect on nephrotoxicity of compd. A. Nephrotoxicity of inhaled compd. A in rats was assocd. with renal uptake of compd. A S-conjugates and cysteine conjugates metab. by renal β-lyase. Manipulation of the β-lyase pathway elicited similar results, whether compd. A was administered by inhalation or i.p. injection. Route of administration does not apparently influence nephrotoxicity of compd. A in rats.145Alauddin, M. M. Positron emission tomography (PET) imaging with 18F-based radiotracers. Am. J. Nucl. Med. Mol. Imaging 2012, 2, 55– 76[PubMed], [CAS], Google Scholar145https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtFOrtb4%253D&md5=55308a81f4916c4a9bf09d92b767c9e6Positron emission tomography (PET) imaging with 18F-based radiotracersAlauddin, Mian M.American Journal of Nuclear Medicine and Molecular Imaging (2012), 2 (1), 55-76CODEN: AJNMAU; ISSN:2160-8407. (e-Century Publishing Corp.)A review. Positron emission tomog. (PET) is a nuclear medicine imaging technique that is widely used in early detection and treatment follow up of many diseases, including cancer. This modality requires positron-emitting isotope labeled biomols., which are synthesized prior to perform imaging studies. Fluorine-18 is one of the several isotopes of fluorine that is routinely used in radiolabeling of biomols. for PET; because of its positron emitting property and favorable half-life of 109.8 min. The biol. active mol. most commonly used for PET is 2-deoxy-2-18F-fluoro-β--glucose (18F-FDG), an analog of glucose, for early detection of tumors. The concns. of tracer accumulation (PET image) demonstrate the metabolic activity of tissues in terms of regional glucose metab. and accumulation. Other tracers are also used in PET to image the tissue concn. In this review, information on fluorination and radiofluorination reactions, radiofluorinating agents, and radiolabeling of various compds. and their application in PET imaging is presented.146Pauwels, E. K. J. 18F-labeled fluorodeoxyglucose for PET imaging: the working mechanism and its clinical implication. Drugs Future 2001, 26, 659– 668, DOI: 10.1358/dof.2001.026.07.858710[Crossref], [CAS], Google Scholar146https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXot1CntL0%253D&md5=f75a1c778a0d193615cedb576573e82a18F-labeled fluorodeoxyglucose for PET imaging: The working mechanism and its clinical implicationPauwels, Ernest K. J.Drugs of the Future (2001), 26 (7), 659-668CODEN: DRFUD4; ISSN:0377-8282. (Prous Science)A review on the uptake mechanism and biochem. processing of Glc and fluorodeoxyglucose (FDG) in normal and malignant cells. Uptake of Glc by normal cells, fate of Glc in normal cells, Glc uptake in tumor cells, hexokinase in tumor cells, and 18F-labeled FDG positron emission tomog. (FDG-PET) are described. The role of FDG-PET in the management of cancer patients is discussed.147Krishnan, H. S.; Ma, L.; Vasdev, N.; Liang, S. H. 18F-Labeling of sensitive biomolecules for positron emission tomography. Chem. - Eur. J. 2017, 23, 15553– 15577, DOI: 10.1002/chem.201701581[Crossref], [PubMed], [CAS], Google Scholar147https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVekt7vP&md5=36ab8ca2d515f0b53ca9ba3eb361b55318F-Labeling of Sensitive Biomolecules for Positron Emission TomographyKrishnan, Hema S.; Ma, Longle; Vasdev, Neil; Liang, Steven H.Chemistry - A European Journal (2017), 23 (62), 15553-15577CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Positron emission tomog. (PET) imaging study of fluorine-18 labeled biomols. is an emerging and rapidly growing area for preclin. and clin. research. The present review focuses on recent advances in radiochem. methods for incorporating fluorine-18 into biomols. via "direct" or "indirect" bioconjugation. Recently developed prosthetic groups and pre-targeting strategies, as well as representative examples in 18F-labeling of biomols. in PET imaging research studies are highlighted.148Lee, K. C.; Lee, S.-Y.; Choe, Y. S.; Chi, D. Y. Metabolic stability of [18F]fluoroalkylbiphenyls. Bull. Korean Chem. Soc. 2004, 25, 1225– 1230, DOI: 10.5012/bkcs.2004.25.8.1225[Crossref], [CAS], Google Scholar148https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXosVKlu7c%253D&md5=be34e3fc985b636ca73833f435ad0204Metabolic stability of [18F]fluoroalkylbiphenylsLee, Kyo Chul; Lee, Sang-Yoon; Choe, Yearn Seong; Chi, Dae YoonBulletin of the Korean Chemical Society (2004), 25 (8), 1225-1230CODEN: BKCSDE; ISSN:0253-2964. (Korean Chemical Society)The stability of fluoroalkyl groups as a pendent on the Ph ring was measured in vitro using rat hepatic microsomes and human serum to predict their in vivo stabilities. We have prepd. three [18F]fluoroalkylbiphenyls as the model compds. of fluoroalkyl arom. compds. to compare the in vitro stabilities. In addn., in vitro stabilities were measured sep. using rat hepatic microsomes and human serum at 37°. Fluoroethylbiphenyl had similar or slightly superior stability to fluoropropylbiphenyl and these two compds. were much more stable than fluoromethylbiphenyl in vitro.149French, A. N.; Napolitano, E.; VanBrocklin, H. F.; Hanson, R. N.; Welch, M. J.; Katzenellenbogen, J. A. Synthesis, radiolabeling and tissue distribution of 11 β-fluoroalkyl- and 11 β-fluoroalkoxy-substituted estrogens: target tissue uptake selectivity and defluorination of a homologous series of fluorine-18-labeled estrogens. Nucl. Med. Biol. 1993, 20, 31– 47, DOI: 10.1016/0969-8051(93)90134-G[Crossref], [PubMed], [CAS], Google Scholar149https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXitVylsr0%253D&md5=51479b76bbfb5f044f99b6ef632d9250Synthesis, radiolabeling and tissue distribution of 11β-fluoroalkyl- and 11β-fluoroalkoxy-substituted estrogens: target tissue uptake selectivity and defluorination of a homologous series of fluorine-18-labeled estrogensFrench, Andrew N.; Napolitano, Elio; Vanbrocklin, Henry F.; Hanson, Robert N.; Welch, Michael J.; Katzenellenbogen, John A.Nuclear Medicine and Biology (1993), 20 (1), 31-47CODEN: NMBIEO; ISSN:0883-2897.Six estrogens substituted at the 11β-position with a fluoroalkyl or fluoroalkoxy substituent were prepd. These compds. bind to the estrogen receptor with moderate to high affinity, with the fluoroalkyl analogs being higher affinity binders than the fluoroalkoxy ones. All of these fluorine-substituted estrogens were prepd. in fluorine-18-labeled form, with high radiochem. purity and at effective specific activities (15.4-50.4 TBq/mmol; 415-1362 Ci/mmol)adequate for biodistribution studies. In immature female rats, 5 of the 6 fluoroestrogens showed selective uptake by the uterus, with uterine uptake as a percent of the injected dose per g being 4-9% at 1 h, and uterus-to-blood or uterus-to-muscle ratios being 10-40. Selective uterine uptake was eliminated by coadministration of a blocking dose of unlabeled estradiol. The only compd. that did not show selective uterine uptake was 11β-fluoropropoxyl estradiol; its rapid metab. and its low affinity for the estrogen receptor, particularly at 25°, may account for its lack of specific uptake. The level of bone activity, an index of metabolic defluorination, shows that the defluorination rates of these six estrogens are a complex function of structure and functionality. The least prone to defluorination is 11β-(2-fluoroethoxy)estradiol and the most prone is 11β-(2-fluoroethyl)estradiol. The extent of defluorination of the remaining compds. shows weak evidence for the protective effect of a heteroatom-substituted beta to the site of metab. (the CH bonds on the fluorine-bearing carbon atom). The binding affinity, tissue distribution and metab. of these 11β-fluoroalkyl- and fluoroalkoxy-substituted estrogens further the understanding of the behavior of fluorine-18-labeled estrogens as potential imaging agents for estrogen receptor-pos. breast cancer.150Irurre, J., Jr.; Casas, J.; Messeguer, A. Resistance of the 2,2,2-trifluoroethoxy aryl moiety to the cytochrome P-450 metabolism in rat liver microsomes. Bioorg. Med. Chem. Lett. 1993, 3, 179– 182, DOI: 10.1016/S0960-894X(01)80872-6[Crossref], [CAS], Google Scholar150https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXktVShsL8%253D&md5=3388a20b7a0ee665e8224535556eef5fResistance of the 2,2,2-trifluoroethoxy aryl moiety to the cytochrome P-450 metabolism in rat liver microsomesIrurre, Josep, Jr.; Casas, Josefina; Messeguer, AngelBioorganic & Medicinal Chemistry Letters (1993), 3 (2), 179-82CODEN: BMCLE8; ISSN:0960-894X.7-(2,2,2-Trifluoroethoxy)coumarin (I) or 4'-(2,2,2-trifluoroethoxy)acetanilide (II), fluorinated analogs of the well known cytochrome P 450 deethylase (EC 1.14.14.1) substrates 7-ethoxycoumarin and phenacetin, resp., remained unaltered after incubation with rat liver microsomes in the presence of NADPH. In addn., compds. I and II showed a moderate activity as inhibitors of the above enzymes. These results suggest that the CF3CH2O- group could play an important role in the design of bioactive compds. when a metabolic resistance at a specific position is desired.151Diana, G. D.; Rudewicz, P.; Pevear, D. C.; Nitz, T. J.; Aldous, S. C.; Aldous, D. J.; Robinson, D. T.; Draper, T.; Dutko, F. J.; Aldi, C.; Gendron, G.; Oglesby, R. C.; Volkots, D. L.; Reumann, M.; Bailey, T. R.; Czernial, R.; Block, T.; Roland, R.; Opperman, J. Picornavirus inhibitors: trifluoromethyl substitution provides a global protective effect against hepatic metabolism. J. Med. Chem. 1995, 38, 1355– 1371, DOI: 10.1021/jm00008a014[ACS Full Text], [CAS], Google Scholar151https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXkvVWqtLs%253D&md5=36a8ac2604f10f57d2aad9b131d84734Picornavirus Inhibitors: Trifluoromethyl Substitution Provides a Global Protective Effect against Hepatic MetabolismDiana, Guy D.; Rudewicz, Patrick; Pevear, Daniel C.; Nitz, Theodore J.; Aldous, Suzanne C.; Aldous, David J.; Robinson, David T.; Draper, Tandy; Dutko, Frank J.; et al.Journal of Medicinal Chemistry (1995), 38 (8), 1355-71CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Several modifications of the oxazoline ring of WIN 54954, a broad spectrum antipicornavirus compd., have been prepd. to address the acid lability and metabolic instability of this compd. The authors have previously shown that the oxadiazole analog (I) displayed comparable activity against a variety of rhinoviruses and appeared to be stable to acid. A monkey liver microsomal assay was developed to examine the metabolic stability in vitro of both compds., and it was detd. that WIN 54954 displayed 18 metabolic products while I was converted to 8 products. Two major products of I were detd. by LC-MS/MS to be monohydroxylated at each of the terminal Me groups. Replacement of the Me on the isoxazole ring with a trifluoromethyl group, while preventing hydroxylation at this position, did not reduce the sensitivity of the mol. to microsomal metab. at other sites. However, the (trifluoromethyl)oxadiazole not only prevented hydroxylation at this position but also provided protection at the isoxazole end of the mol., resulting in only two minor products to the extent of 4%. The major product was identified as the monohydroxylated compd. The global metabolic protective effect of trifluoromethyl group on the oxadiazole ring was further demonstrated by examg. a variety of analogous including heterocyclic replacements of the isoxazole ring. In each case, the trifluoromethyl analog displayed a protective effect when compared to the corresponding Me analog.152Cliffe, I. A. A retrospect on the discovery of WAY-100635 and the prospect for improved 5-HT(1A) receptor PET radioligands. Nucl. Med. Biol. 2000, 27, 441– 447, DOI: 10.1016/S0969-8051(00)00109-8[Crossref], [PubMed], [CAS], Google Scholar152https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXmtVGjt7k%253D&md5=b65fa42e0685aeb1ca0e3e0e6344402dA retrospect on the discovery of way-100635 and the prospect for improved 5-HT1A receptor PET radioligandsCliffe, I. A.Nuclear Medicine and Biology (2000), 27 (5), 441-447CODEN: NMBIEO; ISSN:0969-8051. (Elsevier Science Inc.)A review with 45 refs. An outline is given of the developments that led to the identification of [O-methyl-11C]WAY-100635 (4) as the first useful PET ligand for imaging serotonin1A (5-HT1A) receptors in the living human brain. Recent attempts to develop 5-HT1A receptor radioligands superior to 4 are reviewed, and [carbonyl-11C]WAY-100635 (6) has been shown to be the best currently available radioligand for human studies. Of other 11C-radiolabeled compds., [O-methyl-11C](R,S)-CPC-222 (9), DWAY (8), and [11C]NAD-299 (14) all demonstrate specific binding to 5-HT1A receptors in animals and warrant further expedited studies in humans. The trans-fluorocyclohexane, 12, and fluorobenzene, [18F]p-MPPF 13, are highlighted as examples of promising 18F-labeled ligands.153Pike, V. W.; Halldin, C.; Wikstrom, H.; Marchais, S.; McCarron, J. A.; Sandell, J.; Nowicki, B.; Swahn, C. G.; Osman, S.; Hume, S. P.; Constantinou, M.; Andree, B.; Farde, L. Radioligands for the study of brain 5-HT(1A) receptors in vivo--development of some new analogues of way. Nucl. Med. Biol. 2000, 27, 449– 455, DOI: 10.1016/S0969-8051(00)00110-4[Crossref], [PubMed], [CAS], Google Scholar153https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXmtVGjt7Y%253D&md5=f113308beeb2ea0ee186052477101f45Radioligands for the study of brain 5-HT1A receptors: in vivo development of some new analogues of WAYPike, V. W.; Halldin, C.; Wikstrom, H.; Marchais, S.; McCarron, J. A.; Sandell, J.; Nowicki, B.; Swahn, C.-G.; Osman, S.; Hume, S. P.; Constantinou, M.; Andree, B.; Farde, L.Nuclear Medicine and Biology (2000), 27 (5), 449-455CODEN: NMBIEO; ISSN:0969-8051. (Elsevier Science Inc.)A review with 46 refs. [Carbonyl-11C]WAY-100635 (WAY) has proved to be a very useful radioligand for the imaging of brain 5-HT1A receptors in human brain in vivo with positron emission tomog. (PET). WAY is now being applied widely for clin. research and drug development. However, WAY is rapidly cleared from plasma and is also rapidly metabolized. A comparable radioligand, with a higher and more sustained delivery to brain, is desirable since these properties might lead to better biomathematical modeling of acquired PET data. There are also needs for other types of 5-HT1A receptor radioligands, for example, ligands sensitive to elevated serotonin levels, ligands labeled with longer-lived fluorine-18 for distribution to "satellite" PET centers, and ligands labeled with iodine-123 for single photon emission computerized tomog. (SPECT) imaging. Here we describe our progress toward these aims through the exploration of WAY analogs, including the development of [carbonyl-11C]desmethyl-WAY (DWAY) as a promising, more brain-penetrant radioligand for PET imaging of human 5-HT1A receptors, and (pyridinyl-6-halo)-analogs as promising leads for the development of radiohalogenated ligands.154Al Hussainy, R.; Verbeek, J.; van der Born, D.; Molthoff, C.; Booij, J.; Herscheid, J. K. Synthesis, biodistribution and PET studies in rats of 18F-labeled bridgehead fluoromethyl analogues of WAY-100635. Nucl. Med. Biol. 2012, 39, 1068– 1076, DOI: 10.1016/j.nucmedbio.2012.04.002[Crossref], [PubMed], [CAS], Google Scholar154https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhtlamu7nM&md5=488c8af356c6fd2550723321be5bcb7eSynthesis, biodistribution and PET studies in rats of 18F-Labeled bridgehead fluoromethyl analogues of WAY-100635Al Hussainy, Rana; Verbeek, Joost; van der Born, Dion; Molthoff, Carla; Booij, Jan; Herscheid, J. D. M.Nuclear Medicine and Biology (2012), 39 (7), 1068-1076CODEN: NMBIEO; ISSN:0969-8051. (Elsevier)In vitro screening of fluoromethyl bridge-fused ring (BFR) analogs of WAY-100635 (5a, 5b and 5c) has shown a high binding affinity and a good selectivity for the 5-HT1A receptor. As these compds. were designed to provide PET ligands with high metabolic stability, they are now radiolabeled with fluorine-18 and investigated in vivo. BFR precursors were synthesized and reacted with fluorine-18 in dry MeCN in the presence of 2,2,2-kryptofix and K2CO3. In rats, biodistribution and PET studies were performed using [18F]5a, [18F]5b and [18F]5c. The binding specificity was detd. by administration of non-labeled WAY-100635 prior to the radiolabeled ligands. [18F]5 ligands were synthesized in overall radiochem. yields of 24%-45%, resp. with a radiochem. purity of > 98%. Relatively good hippocampus to cerebellum ratios of 5.55, 4.79 and 5.45, resp. were reached at 45 min pi. However, PET studies indicated defluorination of the radioligands by showing high accumulation of radioactivity in the bones in the order of [18F]5a ≈ [18F]5b > [18F]5c. Also in vivo, the radioligands bind preferentially to the 5-HT1A receptor. Unfortunately, no metabolic stability with regard to defluorination was obsd. in rats.155Saigal, N.; Pichika, R.; Easwaramoorthy, B.; Collins, D.; Christian, B. T.; Shi, B.; Narayanan, T. K.; Potkin, S. G.; Mukherjee, J. Synthesis and biologic evaluation of a novel serotonin 5-HT1A receptor radioligand, 18F-labeled mefway, in rodents and imaging by PET in a nonhuman primate. J. Nucl. Med. 2006, 47, 1697– 1706[PubMed], [CAS], Google Scholar155https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFKjtrbL&md5=69b747cf192da90a097b5c27db14051eSynthesis and biologic evaluation of a novel serotonin 5-HT1A receptor radioligand, 18F-labeled mefway, in rodents and imaging by PET in a nonhuman primateSaigal, Neil; Pichika, Rama; Easwaramoorthy, Balasubramaniam; Collins, Daphne; Christian, Bradley T.; Shi, Bingzhi; Narayanan, Tanjore K.; Potkin, Steven G.; Mukherjee, JogeshwarJournal of Nuclear Medicine (2006), 47 (10), 1697-1706CODEN: JNMEAQ; ISSN:0161-5505. (Society of Nuclear Medicine)Serotonin 5-HT1A receptors have been implicated in disorders of the central nervous system and, therefore, are being studied by PET. Efforts are under way to improve in vivo stability of 5-HT1A agents currently in human use (11C-labeled N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-(2-pyridinyl)cyclohexanecarboxamide [11C-WAY-100635]), 4-(2'-methoxyphenyl)-1-[2'-(N-2''-pyridinyl)-p-18F-fluorobenzamido]ethylpiperazine [18F-MPPF], and 18F-labeled trans-4-fluoro-N-(2-[4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-N-(2-pyridyl)cyclohexanecarboxamide) [18F-FCWAY]. We have synthesized N-{2-[4-(2-methoxyphenyl)piperazinyl]ethyl}-N-(2-pyridyl)-N-(4-18F-fluoromethylcyclohexane)carboxamide (18F-mefway), which contains a 18F on a primary carbon to make the compd. more stable to defluorination. Methods: Radiosynthesis of 18F-mefway was performed in a single tosylate for 18F-fluoride exchange. In vitro binding studies on rat brain slices using 18F-mefway were read on a phosphor imager. Monkey PET studies were performed on a whole-body PET scanner. Results: Binding affinity (inhibitory concn. of 50% [IC50]) of mefway was 26 nmol/L and was comparable to that of WAY-100635, 23 nmol/L. Yields of 18F-mefway were 20%-30% in specific activities of 74-111 GBq/μmol at the end of synthesis. In vitro binding of 18F-mefway in the hippocampus (Hp), colliculus (Co), cortex (Ctx), and other brain regions-with limited binding in the cerebellum (Cer)-was obsd., with ratios of Hp/Cer = 82.3, Co/Cer = 45.8, and Ctx/Cer = 40. Serotonin displaced 18F-mefway from various brain regions with IC50 values in the range of 169-243 nmol/L. PET studies in a rhesus monkey showed 18F-mefway binding in the fontal cortex (FC), temporal cortex (TC) including hippocampus, raphe (Rp), and other brain regions, with ratios of FC/Cer = 9.0, TC/Cer = 10, and Rp/Cer = 3.3. Plasma anal. indicated the presence of approx. 30% of 18F-mefway at 150-180 min after injection. Conclusion: The high ratios in specific brain regions such as the hippocampus suggest that 18F-mefway has potential as a PET agent for 5HT1A receptors.156Rodil, A.; Bosisio, S.; Ayoup, M. S.; Quinn, L.; Cordes, D. B.; Slawin, A. M. Z.; Murphy, C. D.; Michel, J.; O’Hagan, D. Metabolism and hydrophilicity of the polarised ‘Janus face’ all-cis tetrafluorocyclohexyl ring, a candidate motif for drug discovery. Chem. Sci. 2018, 9, 3023– 3028, DOI: 10.1039/C8SC00299A[Crossref], [PubMed], [CAS], Google Scholar156https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXivFSmsr8%253D&md5=d6b676dd05116c6a9e1c39093f415fabMetabolism and hydrophilicity of the polarised 'Janus face' all-cis tetrafluorocyclohexyl ring, a candidate motif for drug discoveryRodil, Andrea; Bosisio, Stefano; Ayoup, Mohammed Salah; Quinn, Laura; Cordes, David B.; Slawin, Alexandra M. Z.; Murphy, Cormac D.; Michel, Julien; O'Hagan, DavidChemical Science (2018), 9 (11), 3023-3028CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)The metab. and polarity of the all-cis tetra-fluorocyclohexane motif is explored in the context of its potential as a motif for inclusion in drug discovery programs. Biotransformations of Ph all-cis tetra-, tri- and di- fluoro cyclohexanes with the human metab. model organism Cunninghamella elegans illustrates various hydroxylated products, but limited to benzylic hydroxylation for the Ph all-cis tetrafluorocyclohexyl ring system. Evaluation of the lipophilicities (log P) indicates a significant and progressive increase in polarity with increasing fluorination on the cyclohexane ring system. Mol. dynamics simulations indicate that water assocs. much more closely with the hydrogen face of these Janus face cyclohexyl rings than the fluorine face owing to enhanced hydrogen bonding interactions with the polarised hydrogens and water.157Harlow, P. H.; Perry, S. J.; Stevens, A. J.; Flemming, A. J. Comparative metabolism of xenobiotic chemicals by cytochrome P450s in the nematode Caenorhabditis elegans. Sci. Rep. 2018, 8, 13333, DOI: 10.1038/s41598-018-31215-w[Crossref], [PubMed], [CAS], Google Scholar157https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3c3mtlCktg%253D%253D&md5=85e0c599a138cbdacc9794815095d92eComparative metabolism of xenobiotic chemicals by cytochrome P450s in the nematode Caenorhabditis elegansHarlow Philippa H; Perry Simon J; Stevens Alexander J; Flemming Anthony JScientific reports (2018), 8 (1), 13333 ISSN:.We investigated the metabolic capabilities of C. elegans using compounds whose metabolism has been well characterised in mammalian systems. We find that similar metabolites are produced in C. elegans as in mammals but that C. elegans is deficient in CYP1-like metabolism, as has been seen in other studies. We show that CYP-34A9, CYP-34A10 and CYP-36A1 are the principal enzymes responsible for the metabolism of tolbutamide in C. elegans. These are related to the mammalian enzymes that metabolise this compound but are not the closest homologs suggesting that sequence comparison alone will not predict functional conservation among cytochrome P450s. In mammals, metabolite production from amytryptiline and dextromethorphan is dependent on specific cytochrome P450s. However, in C. elegans we did not find evidence of similar specificity: the same metabolites were produced but in small amounts by numerous cytochrome P450s. We conclude that, while some aspects of cytochrome P450 mediated metabolism in C. elegans are similar to mammals, there are differences in the production of some metabolites and in the underlying genetics of metabolism.158Huchet, Q. A.; Kuhn, B.; Wagner, B.; Kratochwil, N. A.; Fischer, H.; Kansy, M.; Zimmerli, D.; Carreira, E. M.; Muller, K. Fluorination patterning: A study of structural motifs that impact physicochemical properties of relevance to drug discovery. J. Med. Chem. 2015, 58, 9041– 9060, DOI: 10.1021/acs.jmedchem.5b01455[ACS Full Text], [CAS], Google Scholar158https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhslGmsrzM&md5=20d7a36b75501c02570744857c82f325Fluorination Patterning: A Study of Structural Motifs That Impact Physicochemical Properties of Relevance to Drug DiscoveryHuchet, Quentin A.; Kuhn, Bernd; Wagner, Bjorn; Kratochwil, Nicole A.; Fischer, Holger; Kansy, Manfred; Zimmerli, Daniel; Carreira, Erick M.; Muller, KlausJournal of Medicinal Chemistry (2015), 58 (22), 9041-9060CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The synthesis of a collection of 3-substituted indole derivs. incorporating partially fluorinated Pr and Bu groups is described along with an in-depth study of the effects of various fluorination patterns on their properties, such as lipophilicity, aq. soly., and metabolic stability. The exptl. observations confirm predictions of a marked lipophilicity decrease imparted by a vic-difluoro unit when compared to the gem-difluoro counterparts. The data involving the comparison of the two substitution patterns is expected to benefit mol. design in medicinal chem. and, more broadly, in life as well as materials sciences.159Xie, C.; Gao, X.; Sun, D.; Zhang, Y.; Krausz, K. W.; Qin, X.; Gonzalez, F. J. Metabolic profiling of the novel hypoxia-inducible factor 2α inhibitor PT2385 in vivo and In vitro. Drug Metab. Dispos. 2018, 46, 336– 345, DOI: 10.1124/dmd.117.079723[Crossref], [PubMed], [CAS], Google Scholar159https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpsV2ktL8%253D&md5=c1c2ac8c068be824926381074cc046daMetabolic profiling of the novel hypoxia-inducible factor 2a inhibitor PT2385 in vivo and in vitroXie, Cen; Gao, Xiaoxia; Sun, Dongxue; Zhang, Youbo; Krausz, Kristopher W.; Qin, Xuemei; Gonzalez, Frank J.Drug Metabolism & Disposition (2018), 46 (4), 336-345,S1-S14CODEN: DMDSAI; ISSN:1521-009X. (American Society for Pharmacology and Experimental Therapeutics)PT2385 is a first-in-class, selective small-mol. inhibitor of hypoxia-inducible factor-2α (HIF-2α) developed for the treatment of advanced clear cell renal cell carcinoma. Preclin. results demonstrated that PT2385 has potent antitumor efficacy in mouse xenograft models of kidney cancer. It also has activity toward metabolic disease in a mouse model. However, no metab. data are currently publically available. It is of great importance to characterize the metab. of PT2385 and identify its effect on systemic homeostasis in mice. High-resoln. mass spectrometry-based metabolomics was performed to profile the biotransformation of PT2385 and PT2385-induced changes in endogenous metabolites. Liver microsomes and recombinant drug-metabolizing enzymes were used to det. the mechanism of PT2385 metab. Real-time polymerase chain reaction anal. was employed to investigate the reason for the PT2385-induced bile acid dysregulation. A total of 12 metabolites of PT2385 was characterized, generated from hydroxylation (M1, M2), dihydroxylation and desatn. (M3, M4), oxidative-defluorination (M7), glucuronidation (M8), N-acetylcysteine conjugation (M9), and secondary methylation (M5, M6) and glucuronidation (M10, M11, and M12). CYP2C19 was the major contributor to the formation of M1, M2, and M7, UGT2B17 to M8, and UGT1A1/3 to M10-M12. The bile acid metabolites taurocholic acid and tauro-β-muricholic acid were elevated in serum and liver of mice after PT2385 treatment. Gene expression anal. further revealed that intestinal HIF-2α inhibition by PT2385 treatment upregulated the hepatic expression of CYP7A1, the rate-limiting enzyme in bile acid synthesis. This study provides metabolic data and an important ref. basis for the safety evaluation and rational clin. application of PT2385.160Xu, R.; Wang, K.; Rizzi, J. P.; Huang, H.; Grina, J. A.; Schlachter, S. T.; Wang, B.; Wehn, P. M.; Yang, H.; Dixon, D. D.; Czerwinski, R. M.; Du, X.; Ged, E. L.; Han, G.; Tan, H.; Wong, T.; Xie, S.; Josey, J. A.; Wallace, E. M. 3-[(1S,2S,3R)-2,3-Difluoro-1-hydroxy-7-methylsulfonylindan-4-yl]oxy-5-fluorobenzo nitrile (PT2977), a hypoxia-inducible factor 2α (HIF-2α) inhibitor for the treatment of clear cell renal cell carcinoma. J. Med. Chem. 2019, 62, 6876– 6893, DOI: 10.1021/acs.jmedchem.9b00719[ACS Full Text], [CAS], Google Scholar160https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1ensL%252FN&md5=ec1f0a964e0fda36f92261c30edf284e3-[(1S,2S,3R)-2,3-Difluoro-1-hydroxy-7-methylsulfonylindan-4-yl]oxy-5-fluorobenzonitrile (PT2977), a Hypoxia-Inducible Factor 2α (HIF-2α) Inhibitor for the Treatment of Clear Cell Renal Cell CarcinomaXu, Rui; Wang, Keshi; Rizzi, James P.; Huang, Heli; Grina, Jonas A.; Schlachter, Stephen T.; Wang, Bin; Wehn, Paul M.; Yang, Hanbiao; Dixon, Darryl D.; Czerwinski, Robert M.; Du, Xinlin; Ged, Emily L.; Han, Guangzhou; Tan, Huiling; Wong, Tai; Xie, Shanhai; Josey, John A.; Wallace, Eli M.Journal of Medicinal Chemistry (2019), 62 (15), 6876-6893CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The hypoxia-inducible factor 2α (HIF-2α) is a key oncogenic driver in clear cell renal cell carcinoma (ccRCC). Our first HIF-2α inhibitor PT2385 demonstrated promising proof of concept clin. activity in heavily pretreated advanced ccRCC patients. However, PT2385 was restricted by variable and dose-limited pharmacokinetics resulting from extensive metab. of PT2385 to its glucuronide metabolite. Herein we describe the discovery of second-generation HIF-2α inhibitor PT2977 with increased potency and improved pharmacokinetic profile achieved by redn. of phase 2 metab. Structural modification by changing the geminal difluoro group in PT2385 to a vicinal difluoro group resulted in enhanced potency, decreased lipophilicity, and significantly improved pharmacokinetic properties. In a phase 1 dose-escalation study, the clin. pharmacokinetics for PT2977 supports the hypothesis that attenuating the rate of glucuronidation would improve exposure and reduce variability in patients. Early evidence of clin. activity shows promise for PT2977 in the treatment of ccRCC.161Hughes, S. C.; Shardlow, P. C.; Hollis, F. J.; Scott, R. J.; Motivaras, D. S.; Allen, A.; Rousell, V. M. Metabolism and disposition of fluticasone furoate, an enhanced-affinity glucocorticoid, in humans. Drug Metab. Dispos. 2008, 36, 2337– 2344, DOI: 10.1124/dmd.108.022137[Crossref], [PubMed], [CAS], Google Scholar161https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtlWiu77L&md5=d51c628e6fdc783fa5fd8dc58d4cdaa6Metabolism and disposition of fluticasone furoate, an enhanced-affinity glucocorticoid, in humansHughes, Stephen C.; Shardlow, Peter C.; Hollis, Frank J.; Scott, Rebecca J.; Motivaras, Dimple S.; Allen, Ann; Rousell, Victoria M.Drug Metabolism and Disposition (2008), 36 (11), 2337-2344CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)The purpose of this study was to investigate the metab. and disposition of fluticasone furoate, an enhanced-affinity glucocorticoid receptor agonist, in humans. In a two-part, open-label design study, five healthy male subjects received a p.o. dose of 2 mg of [14C]fluticasone furoate, followed 4 wk later by an i.v. dose of 0.25 mg of [14C]fluticasone furoate (as a 30-min infusion). Oral absorption was rapid and estd. at approx. 30%, although the oral bioavailability was markedly lower at 1.6%, limited by extensive first-pass metab. Plasma clearance was 58.3 L/h, with a vol. of distribution of 642 L and a terminal elimination half-life of 15.3 h. The major circulating component identified in plasma exts. after i.v. and p.o. dosing was unchanged parent compd., with 17β-carboxylic acid (GW 694301X) also being notable after p.o. administration. Mean recovery of radioactivity was approx. 92 and 102% at 216 and 168 h after i.v. and p.o. administration, resp., with most (at least 90%) recovered in the feces. Fluticasone furoate was extensively metabolized, with only trace amts. of unchanged parent compd. obsd. in feces following either route of administration. The predominant pathway was removal of the S-fluoromethyl carbothioate group to yield GW 694301X. Other pathways included oxidative defluorination to yield a hydroxyl at the C6 position. There was no evidence for metabolic loss of the furoate group from fluticasone furoate or any of its metabolites. Evidence presented suggests that enterocytes have a role in the metab. of unabsorbed fluticasone furoate.162Teitelbaum, P. J.; Chu, N. I.; Cho, D.; Tökés, L.; Patterson, J. W.; Wagner, P. J.; Chaplin, M. D. Mechanism for the oxidative defluorination of flunisolide. J. Pharmacol. Exp. Ther. 1981, 218, 16– 22[PubMed], [CAS], Google Scholar162https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXkslCisL8%253D&md5=5aeb5f0bda2f31d237bfccd176025878Mechanism for the oxidative defluorination of flunisolideTeitelbaum, Philip J.; Chu, Nancy I.; Cho, Diane; Toekes, Laszlo; Patterson, John W.; Wagner, Paul J.; Chaplin, Melvin D.Journal of Pharmacology and Experimental Therapeutics (1981), 218 (1), 16-22CODEN: JPETAB; ISSN:0022-3565.Flunisolide (I) [3385-03-3] was converted to 6β,11β,16α,17α,21-pentahydroxypregna-1,4-diene-3,20-dione-16,17-acetonide (6β-OH metabolite) [73603-90-4] by mouse liver microsomes, but no activity was obsd. with mouse lung, intestine or kidney microsomes. Two addnl. metabolites of I also formed by mouse hepatic microsomes were identified by mass spectral anal. to be 11β,16α,17α,21-tetrahydroxypregna-1,4-diene-3,6,20-trione 16,17-acetonide (6-keto metabolite) [78228-98-5] and Δ6-flunisolide [78245-15-5]. The formation of all 3 metabolites required NADPH [53-57-6], was inhibited by CO, and was stimulated by pretreating mice with phenobarbital. A time-course study suggested the 6-keto metabolite was an intermediate in the formation of the 6β-OH metabolite. When added to microsomes, the 6-keto metabolite was converted to the 6β-OH metabolite by a CO-insensitive enzyme. Apparently, the conversion of I to the 6β-OH metabolite is catalyzed by a multi-enzyme pathway via a stable intermediate, the 6-keto metabolite. The initial reaction which leads to the formation of the 6-keto metabolite is catalyzed by a cytochrome P-450-mediated microsomal monoxygenase(s), but the redn. of the 6-keto metabolite to the 6β-OH metabolite is cytochrome P-450-independent.163Moore, C. D.; Roberts, J. K.; Orton, C. R.; Murai, T.; Fidler, T. P.; Reilly, C. A.; Ward, R. M.; Yost, G. S. Metabolic pathways of inhaled glucocorticoids by the CYP3A enzymes. Drug Metab. Dispos. 2013, 41, 379– 389, DOI: 10.1124/dmd.112.046318[Crossref], [PubMed], [CAS], Google Scholar163https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXitlGis7k%253D&md5=e89d33f0da338a12b5258e543ae6234fMetabolic pathways of inhaled glucocorticoids by the CYP3A enzymesMoore, Chad D.; Roberts, Jessica K.; Orton, Christopher R.; Murai, Takahiro; Fidler, Trevor P.; Reilly, Christopher A.; Ward, Robert M.; Yost, Garold S.Drug Metabolism & Disposition (2013), 41 (2), 379-389CODEN: DMDSAI; ISSN:1521-009X. (American Society for Pharmacology and Experimental Therapeutics)Asthma is one of the most prevalent diseases in the world, for which the mainstay treatment has been inhaled glucocorticoids (GCs). Despite the widespread use of these drugs, approx. 30% of asthma sufferers exhibit some degree of steroid insensitivity or are refractory to inhaled GCs. One hypothesis to explain this phenomenon is interpatient variability in the clearance of these compds. The objective of this research is to det. how metab. of GCs by the CYP3A family of enzymes could affect their effectiveness in asthmatic patients. In this work, the metab. of four frequently prescribed inhaled GCs, triamcinolone acetonide, flunisolide, budesonide, and fluticasone propionate, by the CYP3A family of enzymes was studied to identify differences in their rates of clearance and to identify their metabolites. Both interenzyme and interdrug variability in rates of metab. and metabolic fate were obsd. CYP3A4 was the most efficient metabolic catalyst for all the compds., and CYP3A7 had the slowest rates. CYP3A5, which is particularly relevant to GC metab. in the lungs, was also shown to efficiently metabolize triamcinolone acetonide, budesonide, and fluticasone propionate. In contrast, flunisolide was only metabolized via CYP3A4, with no significant turnover by CYP3A5 or CYP3A7. Common metabolites included 6β-hydroxylation and Δ6-dehydrogenation for triamcinolone acetonide, budesonide, and flunisolide. The structure of Δ6-flunisolide was unambiguously established by NMR anal. Metab. also occurred on the D-ring substituents, including the 21-carboxy metabolites for triamcinolone acetonide and flunisolide. The novel metabolite 21-nortriamcinolone acetonide was also identified by liq. chromatog.-mass spectrometry and NMR anal.164Biggadike, K.; Bledsoe, R. K.; Hassell, A. M.; Kirk, B. E.; McLay, I. M.; Shewchuk, L. M.; Stewart, E. L. X-ray crystal structure of the novel enhanced-affinity glucocorticoid agonist fluticasone furoate in the glucocorticoid receptor-ligand binding domain. J. Med. Chem. 2008, 51, 3349– 3352, DOI: 10.1021/jm800279t[ACS Full Text], [CAS], Google Scholar164https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXms1Gis7w%253D&md5=bb9838014ade0fbe90c0693b3a8191eaX-ray Crystal Structure of the Novel Enhanced-Affinity Glucocorticoid Agonist Fluticasone Furoate in the Glucocorticoid Receptor-Ligand Binding DomainBiggadike, Keith; Bledsoe, Randy K.; Hassell, Anne M.; Kirk, Barrie E.; McLay, Iain M.; Shewchuk, Lisa M.; Stewart, Eugene L.Journal of Medicinal Chemistry (2008), 51 (12), 3349-3352CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)An X-ray crystal structure is reported for the novel enhanced-affinity glucocorticoid agonist fluticasone furoate (FF) in the ligand binding domain of the glucocorticoid receptor. Comparison of this structure with those of dexamethasone and fluticasone propionate shows the 17α furoate ester to occupy more fully the lipophilic 17α pocket on the receptor, which may account for the enhanced glucocorticoid receptor binding of FF.165Krauser, J. A.; Guengerich, F. P. Cytochrome P450 3A4-catalyzed testosterone 6β-hydroxylation stereochemistry, kinetic deuterium isotope effects, and rate-limiting steps. J. Biol. Chem. 2005, 280, 19496– 19506, DOI: 10.1074/jbc.M501854200[Crossref], [PubMed], [CAS], Google Scholar165https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXktFymsLY%253D&md5=96020ed1c61b61e6147e30b793e39f04Cytochrome P450 3A4-catalyzed Testosterone 6β-Hydroxylation Stereochemistry, Kinetic Deuterium Isotope Effects, and Rate-limiting StepsKrauser, Joel A.; Guengerich, F. PeterJournal of Biological Chemistry (2005), 280 (20), 19496-19506CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Testosterone 6β-hydroxylation is a prototypic reaction of cytochrome P 450 (P 450) 3A4, the major human P 450. Biomimetic reactions produced a variety of testosterone oxidn. products with 6β-hydroxylation being only a minor reaction, indicating that P 450 3A4 has considerable control over the course of steroid hydroxylation because 6β-hydroxylation is not dominant in a thermodynamically controlled oxidn. of the substrate. Several isotopically labeled testosterone substrates were prepd. and used to probe the catalytic mechanism of P 450 3A4: (1) 2,2,4,6,6-2H5; (2) 6,6-2H2; (3) 6α-2H; (4) 6β-2H; and (5) 6β-3H testosterone. Only the 6β-hydrogen was removed by P 450 3A4 and not the 6α, indicating that P 450 3A4 abstrs. hydrogen and rebounds oxygen only at the β face. Anal. of the rates of hydroxylation of 6β-1H-, 6β-2H-, and 6β-3H-labeled testosterone and application of the Northrop method yielded an apparent intrinsic kinetic deuterium isotope effect (Dk) of 15. The deuterium isotope effects on kcat and kcat/Km in non-competitive reactions were only 2-3. Some switching to other hydroxylations occurred because of 6β-2H substitution. The high Dk value is consistent with an initial hydrogen atom abstraction reaction. Attenuation of the high Dk in the non-competitive expts. implies that C-H bond breaking is not a dominant rate-limiting step. Considerable attenuation of a high Dk value was also seen with a slower P 450 3A4 reaction, the O-dealkylation of 7-benzyloxyquinoline. Thus P 450 3A4 is an enzyme with regioselective flexibility but also considerable regioselectivity and stereoselectivity in product formation, not necessarily dominated by the ease of C-H bond breaking.166Liu, X.; Poddar, S.; Song, L.; Hendrickson, H.; Zhang, X.; Yuan, Y.; Zhou, D.; Zheng, G. Synthesis and liver microsomal metabolic stability studies of a fluoro-substituted δ-tocotrienol derivative. ChemMedChem 2020, DOI: 10.1002/cmdc.201900676167Chang, W.; Mosley, R. T.; Bansal, S.; Keilman, M.; Lam, A. M.; Furman, P. A.; Otto, M. J.; Sofia, M. J. Inhibition of hepatitis C virus NS5A by fluoro-olefin based γ-turn mimetics. Bioorg. Med. Chem. 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Efforts to reproduce these γ-turn-like substructures provided a novel NS5A inhibitor based on a fluoro-olefin isostere. This fluoro-olefin contg. inhibitor exhibited picomolar activity (EC50 = 79 pM) against HCV genotype 1b replicon without measurable cytotoxicity. This level of activity is comparable to the natural peptide-based inhibitors currently under clinic evaluation, and demonstrates that a peptidomimetic approach can serve as a useful strategy to produce potent and structurally unique inhibitors of HCV NS5A.168Bartlett, P. A.; Otake, A. Fluoroalkenes as peptide isosteres: ground state analogs inhibitors of thermolysin. J. Org. 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The correlation indicates that these inhibitors bind as ground state analogs and represent the first direct assessment of the fluoroalkene unit as a peptide surrogate.169Welch, J. T.; Allmendinger, T. Fluoroolefin isosteres. Methods Mol. Med. 1998, 23, 357– 384, DOI: 10.1385/0-89603-517-4:357170Nielsen, O. J.; Javadi, M. S.; Sulbaek Andersen, M. P.; Hurley, M. D.; Wallington, T. J.; Singh, R. Atmospheric chemistry of CF3CF = CH2: Kinetics and mechanisms of gas-phase reactions with Cl atoms, OH radicals, and O3. Chem. Phys. Lett. 2007, 439, 18– 22, DOI: 10.1016/j.cplett.2007.03.053[Crossref], [CAS], Google Scholar170https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXkslWrtL8%253D&md5=e37616d9e52e32cb36d04e670ba7170dAtmospheric chemistry of CF3CF=CH2: Kinetics and mechanisms of gas-phase reactions with Cl atoms, OH radicals, and O3Nielsen, O. J.; Javadi, M. S.; Sulbaek Andersen, M. P.; Hurley, M. D.; Wallington, T. J.; Singh, R.Chemical Physics Letters (2007), 439 (1-3), 18-22CODEN: CHPLBC; ISSN:0009-2614. (Elsevier B.V.)Long path length FTIR-smog chamber techniques were used to det. k(Cl + CF3CF=CH2) = (7.03 ± 0.59) × 10-11, k(OH + CF3CF=CH2) = (1.05 ± 0.17) × 10-12, and k(O3 + CF3CF=>CH2) = (2.77 ± 0.21) × 10-21 cm3 mol.-1 s-1 in 700 Torr of N2, N2/O2, or air diluent at 296 K. CF3CF=CH2 has an atm. lifetime of approx. 11 days and a global warming potential (100 yr time horizon) of four. CF3CF=CH2 has a negligible global warming potential and will not make any significant contribution to radiative forcing of climate change.171Tang, X.; Madronich, S.; Wallington, T.; Calamari, D. Changes in tropospheric composition and air quality. J. Photochem. 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Assuming that other factors remain const., addnl. UV-B will increase the rate at which primary pollutants are removed from the troposphere. Increased UV-B is expected to increase the concn. of hydroxyl radicals and result in faster removal of pollutants such as carbon monoxide, methane, nonmethane hydrocarbons (NMHCs), sulfur oxides, nitrogen oxides, hydrochlorofluorocarbons (HCFCs), and hydrofluorocarbons (HFCs). Concns. of peroxy radicals (both inorg. and org.) are expected to increase, leading to higher atm. levels of hydrogen peroxide and org. peroxides. The effects of UV-B increases on tropospheric O3, OH, methane, CO, and possibly other tropospheric constituents, while not negligible, will be difficult to detect because the concns. of these species are also influenced by many other variable factors (e.g., emissions). Trifluoroacetic acid (TFA) is produced in the atm. by the degrdn. of HCFC-123 (CF3CHCl2), HCFC-124 (CF3CHFCl), and HFC-134a (CF3CH2F), which are used as substitutes for ozone-depleting substances. The atm. oxidn. mechanisms of these replacement compds. are well established. Reported measurements of TFA in rain, rivers, lakes, and oceans show it to be a ubiquitous component of the hydrosphere, present at levels much higher than can be explained by reported sources. The levels of TFA produced by the atm. degrdn. of HFCs and HCFCs emitted up to the year 2020 are estd. to be orders of magnitude below those of concern, and to make only a minor contribution to the current environmental burden of TFA. No significant effects on humans or the environment have been identified from TFA produced by atm. degrdn. of HCFCs and HFCs. Numerous std. short-term studies have shown that TFA has, at most, moderate toxicity.172Cook, E. W.; Pierce, J. S. Toxicology of fluoro-olefins. Nature 1973, 242, 337– 338, DOI: 10.1038/242337a0[Crossref], [PubMed], [CAS], Google Scholar172https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3sXhsVCjtrY%253D&md5=73681c1675dfb3f05bff704bb3d266e1Toxicology of fluoroolefinsCook, E. W.; Pierce, J. S.Nature (London, United Kingdom) (1973), 242 (5396), 337-8CODEN: NATUAS; ISSN:0028-0836.A review with 10 refs. The direct correlation between the toxicity of fluoroolefins and the reactivity of the olefins to nucleophiles, and the enhancement of the toxicity by olefin hydrolysis are discussed.173Timperley, C. M. Fluoroalkene chemistry. Part 1. Highly-toxic fluorobutenes and their mode of toxicity: reactions of perfluoroisobutene and polyfluorinated cyclobutenes with thiols. J. Fluorine Chem. 2004, 125, 685– 693, DOI: 10.1016/j.jfluchem.2003.11.021[Crossref], [CAS], Google Scholar173https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXjvVGgsbw%253D&md5=e73daba9789a18af94ebfcbf01f741cbFluoroalkene chemistry. Part 1. Highly-toxic fluorobutenes and their mode of toxicity: reactions of perfluoroisobutene and polyfluorinated cyclobutenes with thiolsTimperley, Christopher M.Journal of Fluorine Chemistry (2004), 125 (5), 685-693CODEN: JFLCAR; ISSN:0022-1139. (Elsevier Science B.V.)The reactions of four highly-toxic fluorobutenes - perfluoroisobutene (PFIB), 1-hydropentafluorocyclobutene (1-H), hexafluorocyclobutene (HFCB) and 3-chloropentafluorocyclobutene (3-Cl)-with propanethiol, 2,6-dimethoxybenzenethiol and N-acetylcysteine iso-Pr ester were studied. PFIB and HFCB reacted with two molar equivalents of the aliph. thiols, but with only one molar equivalent of the arom. thiol (presumably due to steric hindrance) and resembled phosgene in their reactivity. The fluorocyclobutenes 1-H and 3-Cl reacted with one and up to three molar equivalents of the aliph. thiols, resp., but with only one molar equivalent of the arom. thiol. The products of allyl and vinyl substitution were isolated and characterized as fully as possible. The inhalation toxicities of the fluorocyclobutenes to rodents correlated with the no. of easily-displaceable fluorine substituents, supporting the contention that toxicity is due to reaction with biol. thiols in the lung.174Timperley, C. M. Fluoroalkene chemistry. Part 2. Reactions of thiols with some toxic 1,2-dichlorinatedpolyfluorocycloalkenes. J. Fluorine Chem. 2004, 125, 1265– 1272, DOI: 10.1016/j.jfluchem.2004.02.009[Crossref], [CAS], Google Scholar174https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmvV2htbk%253D&md5=9dfd13f88e44585d38ef48a147005514Fluoroalkene chemistry Part 2. Reactions of thiols with some toxic 1,2-dichlorinated polyfluorocycloalkenesTimperley, Christopher M.Journal of Fluorine Chemistry (2004), 125 (9), 1265-1272CODEN: JFLCAR; ISSN:0022-1139. (Elsevier B.V.)1,2-Dichlorotetrafluorocyclobutene, 1,2-dichlorohexafluorocyclopentene and 1,2-dichlorooctafluorocyclohexene were treated with an equimolar amt. of benzenethiol, 2-methoxybenzenethiol, 3-methoxybenzenethiol and 4-methoxybenzenethiol in acetonitrile with potassium carbonate. Each combination of fluoroalkene and thiol gave a mixt. of mono and bis vinyl substitution products whose proportions depended on the ring size of the fluorocycloalkene and the size and electronic characteristics of the thiol. Treatment of 1,2-dichlorotetrafluorocyclobutene with one or two molar equivalents of N-acetylcysteine iso-Pr ester in acetonitrile with potassium carbonate produced the mono and bis vinyl substitution products accordingly. The results support the contention that the high inhalation toxicity of the fluorocycloalkenes is due to reaction with two molar equivalents of biol. thiols in the lung.175Alaaeddin, M. H.; Sapuan, S. M.; Zuhri, M. Y. M.; Zainudin, E. S.; Al-Oqla, F. M. Properties and common industrial applications of polyvinyl fluoride (PVF) and polyvinylidene fluoride (PVDF). IOP Conf. Ser.: Mater. Sci. Eng. 2018, 409, 012021, DOI: 10.1088/1757-899X/409/1/012021[Crossref], [CAS], Google Scholar175https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisFKktrnK&md5=68fc4def1f324630f9984732324a10a4Properties and common industrial applications of polyvinyl fluoride (PVF) and polyvinylidene fluoride (PVDF)Alaaeddin, M. H.; Sapuan, S. M.; Zuhri, M. Y. M.; Zainudin, E. S.; Al-Oqla, Faris M.IOP Conference Series: Materials Science and Engineering (2018), 409 (3rd International Conference on Manufacturing, Material and Metallurgical Engineering, 2018), 012021/1-012021/7CODEN: ICSMGW; ISSN:1757-899X. (IOP Publishing Ltd.)This work features the properties and the common industrial applications of two thermoplastic polymers which are the Polyvinyl fluoride (PVF) and the Polyvinylidene fluoride (PVDF), the polymn. and the manufg. processes of their monomers have been emphasized in this work. It is believed that these two materials are having a no. of shared properties and applications. Their distinguishing characteristics allow them to be utilized in various applications with immense interest to the industrial world. They excel, converge, and slightly diverge in most of their properties with diverse transition phases as well as piezoelec. and pyroelec. effects. Their eminent properties qualify them to be used in a no. of industrial and outdoor applications e.g. insulations, sensing materials, laminations, encapsulations, coatings, membranes, biomaterials, aircraft interiors and photovoltaic applications. In conclusion, this work recommends further in-depth anal. to investigate the correlations between these two polymers and to provide oriented numerical information on their performance.176Report on Carcinogens Background Document for Vinyl Fluoride; Technology Planning and Management Corporation: Durham, NC, 2000. https://ntp.niehs.nih.gov/ntp/newhomeroc/roc10/vf_no_appendices_508.pdf (accessed 2019-11-02).177Kennedy, G. L., Jr. Toxicology of fluorine-containing monomers. Crit. Rev. Toxicol. 1990, 21, 149– 170, DOI: 10.3109/10408449009089877[Crossref], [PubMed], [CAS], Google Scholar177https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXksVemur4%253D&md5=944bfb73a39d10f1119fd04673b924d3Toxicology of fluorine-containing monomersKennedy, G. L., Jr.Critical Reviews in Toxicology (1990), 21 (2), 149-70CODEN: CRTXB2; ISSN:0045-6446.A review with 144 refs. on the toxicol. of bromotrifluroethylene, chlorotrifluoroethylene, hexafluoroacetone, hexafluoroisobutylene, hexafluoropropylene, perfluorobutylene, tetrafluoroethylene, trichloropropene, vinyl fluoride and vinylidine fluoride.178Vinyl Halides (Selected). In Report on Carcinogens, 14th ed.; U.S. Department of Health and Human Services National Toxicology Program, 2016. https://ntp.niehs.nih.gov/ntp/roc/content/profiles/vinylhalides.pdf (accessed 2019-11-02).179IARC Monographs on the Evaluation of Carginogenic Risks to Humans. http://publications.iarc.fr/115 (accessed 2019-11-11).180Bolt, H. M.; Bartsch, H.; Barbin, A. Roles of etheno-DNA adducts in tumorigenicity of olefins. CRC Crit. Rev. Toxicol. 1988, 18, 299– 309, DOI: 10.3109/10408448809037469[Crossref], [CAS], Google Scholar180https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXit1ag&md5=9621c5df595f30a3540b894809d00317Roles of etheno-DNA adducts in the tumorigenicity of olefinsBolt, Hermann M.Critical Reviews in Toxicology (1988), 18 (4), 299-309CODEN: CRTXB2; ISSN:0045-6446.A review with 61 refs. on conditions under which etheno-DNA adducts are formed in vivo and their possible biol. activities.181Ballering, L. A.; Nivard, M. J.; Vogel, E. W. Characterization by two-endpoint comparisons of the genetic toxicity profiles of vinyl chloride and related etheno-adduct forming carcinogens in Drosophila. Carcinogenesis 1996, 17, 1083– 1092, DOI: 10.1093/carcin/17.5.1083[Crossref], [PubMed], [CAS], Google Scholar181https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xjt1KltLY%253D&md5=022ee23845bd6afd02280d13c5042872Characterization by two-endpoint comparisons of the genetic toxicity profiles of vinyl chloride and related etheno-adduct forming carcinogens in DrosophilaBallering, L. A. P.; Nivard, M. J. M.; Vogel, E. W.Carcinogenesis (1996), 17 (5), 1083-1092CODEN: CRNGDP; ISSN:0143-3334. (Oxford University Press)The genetic toxicity profiles of vinyl chloride (VCl), vinyl bromide (VBr), Et carbamate (EC), vinyl carbamate (VC) and some structurally related chems. were investigated in both somatic and germ cells of Drosophila melanogaster. In the white/white+ eye mosaic assay, a screening system measuring predominantly homologous recombination in somatic cells, only marginal genotoxic activities were obsd. for acetyl chloride (ACl), glycol aldehyde (GCA), 2,2'-dichlorodiethyl ether (DDE) and Me carbamate (MC), whereas VCl. 2-Chloroacetaldehyde (CAA), VBr, 2-bromoacetaldehyde (BAA) and EC were clearly recombinogenic in the assay. Those chems. proven to be recombinogenic in somatic cells were investigated further in postmeiotic male germ cells, utilizing as descriptors of their genotoxicity ICL/RL and Mexr-/Mexr+ indexes. The ICL/RL index is the rate of induced chromosome loss (CL), a clastogenic event, dividend by the forward mutation rate, measured as recessive lethal (RL) mutations in 700 loci of the X-chromosome. The Mexr-/Mexr+ mutation enhancement ratio is obtained by detg. RL under excision repair deficient vs. repair proficient conditions. With ICL/RL values (2.7-6.9) similar to those obtained for crosslinking agents, vinyl chloride, vinyl bromide, Et carbamate and vinyl carbamate are all efficient clastogenic agents in Drosophila germ cells. In the absence of excision repair, however, neither CEO nor CAA gave a hyper-mutability response (Mexr-/Mexr+ ≈1). By contrast, VCl, VBr, EC and VC showed clearly enhanced Mexr-/Mexr+ ratios, suggesting that these compds. produce some repairable DNA modification(s) that are not generated by their epoxides. This unexpected finding points to the formation of other, yet unknown, metabolites of vinyl chloride, vinyl bromide, Et carbamate and vinyl carbamate. Our results support the concept that the epoxides chloroethylene oxide (CEO), bromoethylene oxide (BEO) and vinyl carbamate epoxide (VCO) are the most essential mutagenic intermediates. Compared to chloroethylene oxide (CEO), 2-chloroacetaldehyde (CAA) was approx. 50 times less effective in the induction of RL, whereas BAA was inactive as a mutagen. These findings are consistent with the general view that CAA and BAA play no major role in the genotoxic action of vinyl halides.182Cantoreggi, S.; Keller, D. A. Pharmacokinetics and metabolism of vinyl fluoride in vivo and in vitro. Toxicol. Appl. Pharmacol. 1997, 143, 130– 139, DOI: 10.1006/taap.1996.8041[Crossref], [PubMed], [CAS], Google Scholar182https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXhvVSjsLs%253D&md5=7832d84167caf91f3187e0443a046f6dPharmacokinetics and metabolism of vinyl fluoride in vivo and in vitroCantoreggi, Sergio; Keller, Douglas A.Toxicology and Applied Pharmacology (1997), 143 (1), 130-139CODEN: TXAPA9; ISSN:0041-008X. (Academic)In a first set of expts., groups of three rats or five mice were exposed to vinyl fluoride (VF) in a closed-chamber gas uptake system at starting concns. ranging from 50 to 250 ppm. Chamber concns. of VF were measured every 10-12 min by gas chromatog. Partition coeffs. were detd. by the vial equilibration technique and used as parameters for a physiol. based pharmacokinetic (PBPK) model. Mice showed a higher whole-body metabolic capacity compared to rats (Vmaxc = 0.3 vs. 0.1 mg/h-kg). Both species had an estd. Km of ≤0.02 mg/L. The specificity for the oxidn. of VF in vivo was detd. by selective inhibition or induction of CYP 2E1. Inhibition with 4-methylpyrazole completely impaired VF uptake in rats and mice, whereas induction with ethanol (rats only) increased the metabolic capacity by two- to threefold. The pharmacokinetics of VF were also investigated in vitro. Microsomes from rat and mouse liver were incubated in a sealed vial with VF and an NADPH-regenerating system. Headspace concns. (10-300 ppm) were monitored over time by gas chromatog. Consistent with the in vivo data, VF was metabolized faster by mouse microsomes than by rat microsomes (Vmax = 3.5 and 1.1 nmol/h-mg protein, resp.). The rates of metab. by human liver microsomes were generally in the same range as those found with rat liver microsomes (Vmax = 0.5-1.3 nmol/h-mg protein), but one sample was similar to mice (Vmax = 3.3 nmol/h-mg protein). Metabolic rates in human microsomes were found to correlate with the amt. of CYP 2E1 as detd. by Western blotting and by chlorzoxazone 6-hydroxylation. It is concluded that the greater metabolic capacity of mice for VF both in vivo and in vitro may contribute to their greater susceptibility to tumor formation. CYP 2E1 is clearly the main isoenzyme involved in the oxidn. of VF in all species tested. VF pharmacokinetics and metab. in humans may depend upon the interindividual variability in the expression level of CYP 2E1. The excellent correspondence between in vivo and in vitro kinetics in rodents improves substantially the degree of confidence for human in vivo predictions from in vitro data.183Conolly, R. B.; Jaeger, R. J. Acute hepatotoxicity of ethylene and halogenated ethylenes after PCB pretreatment. Environ. Health Perspect. 1977, 21, 131– 135, DOI: 10.1289/ehp.7721131[Crossref], [PubMed], [CAS], Google Scholar183https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXktlOms74%253D&md5=0d3a5c6253e51f2e3bc50928754d8d24Acute hepatotoxicity of ethylene and halogenated ethylenes after PCB pretreatmentConolly, Rory B.; Jaeger, Rudolph J.Environmental Health Perspectives (1977), 21 (), 131-5CODEN: EVHPAZ; ISSN:0091-6765.In rats pretreated with Aroclor 1254 [11097-69-1] (300 μmol/kg, by gavage) inhalation of vinyl chloride [75-01-4] (30,000 ppm) or ethylene [74-85-1] (20,000 ppm), for 4 h, increased serum alanine-α-ketoglutarate transaminase (SAKT) and sorbitol dehydrogenase, resp. within the 8th and the 24th h after exposure was obsd. Exposure temps. of 12.1-30.3° did not affect the hepatotoxicity of the gases; however, at 33.8° both the elevation of SAKT and the mortality were markedly increased. Overnight fasting prior to treatment with vinyl chloride or ethylene increased hepatotoxicity as evidenced by markedly increased sorbitol dehydrogenase. In fasted rats pretreated with Aroclor 1254 and given trichloropropane epoxide (TE) [3083-23-6] a synergistic increase in the toxicity of ethylene and vinyl chloride was obsd. Pretreatment with TE and fasting increased the toxicity of vinyl bromide [593-60-2] while TE, but not fasting, enhanced vinyl fluoride [75-02-5] toxicity. The acute toxicity of the tested compds. may be mediated through epoxide intermediates.184Bolt, H. M.; Peter, H.; Fost, U. Analysis of macromolecular ethylene oxide adducts. Int. Arch. Occup. Environ. Health 1988, 60, 141– 144, DOI: 10.1007/BF00378688[Crossref], [PubMed], [CAS], Google Scholar184https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXlt1ers7k%253D&md5=33d39e8f51888b3f274a70b494cc9b3eAnalysis of macromolecular ethylene oxide adductsBolt, Hermann M.; Peter, Hans; Foest, UlrichInternational Archives of Occupational and Environmental Health (1988), 60 (3), 141-4CODEN: IAEHDW; ISSN:0340-0131.A review with 26 refs. on the formation of macromol. adducts between ethylene oxide and proteins and nucleic acids and methods for their detection.185La, D. K.; Swenberg, J. A. DNA adducts: biological markers of exposure and potential applications to risk assessment. Mutat. Res., Rev. Genet. Toxicol. 1996, 365, 129– 146, DOI: 10.1016/S0165-1110(96)90017-2[Crossref], [PubMed], [CAS], Google Scholar185https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XlvVeitbo%253D&md5=c7acf596e5bf3bc4fc2437f85b2f5f39DNA adducts: biological markers of exposure and potential applications to risk assessmentLa, David K.; Swenberg, James A.Mutation Research, Reviews in Genetic Toxicology (1996), 365 (1-3), 129-146CODEN: MRRTEP; ISSN:0165-1110. (Elsevier B.V.)A review and discussion with many refs. Specificity of DNA adduct formation, relevance of DNA adducts, mutational spectra, DNA repair, current methodologies to measure DNA adducts, quant. relationships between exposure and DNA adducts, potential applications of mol. dosimetry for risk assessment, interspecies extrapolation, etc., are discussed.186Guengerich, F. P. Mechanisms of formation of DNA adducts from ethylene dihalides, vinyl halides, and arylamines. Drug Metab. Rev. 1994, 26, 47– 66, DOI: 10.3109/03602539409029784[Crossref], [PubMed], [CAS], Google Scholar186https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXkslGgtbg%253D&md5=8acb1ad53181eb95905adb52d4bcfaceMechanisms of formation of DNA adducts from ethylene dihalides, vinyl halides, and arylaminesGuengerich, F. PeterDrug Metabolism Reviews (1994), 26 (1-2), 47-66CODEN: DMTRAR; ISSN:0360-2532.A review with 61 refs. This article reviews some studies done in the author's lab. on 3 important classes of carcinogens: ethylene dihalides, vinyl halides, and arylamines.187Swenberg, J. A.; La, D. K.; Scheller, N. A.; Wu, K. Y. Dose-response relationships for carcinogens. Toxicol. Lett. 1995, 82–83, 751– 756, DOI: 10.1016/0378-4274(95)03593-1[Crossref], [PubMed], [CAS], Google Scholar187https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xht1Wrur0%253D&md5=ca3bbcd3d065f7d1fa8a2e2c6a27c565Dose-response relationships for carcinogensSwenberg, James A.; La, David K.; Scheller, Nova A.; Wu, Kuen-yuhToxicology Letters (1995), 82/83 (1-6), 751-6CODEN: TOLED5; ISSN:0378-4274. (Elsevier)A review and discussion with 15 refs. Biotransformation of chem. carcinogens involves both metabolic activation and detoxication. The mol. dose present on DNA as adducts represents a balance between these 2 pathways (formation) and DNA repair. All of these are enzymic processes subject to satn. When none of the pathways is satd., linear mol. dosimetry is expected whereas if metabolic activation is satd., a supralinear response occurs. If detoxication or DNA repair is satd., a sublinear response occurs. With chronic exposure, steady-state concns. of DNA adducts develop and these follow the same patterns. With several alkylating agents, multiple adducts are formed. The extent of formation is chem. defined, but different DNA repair pathways can be involved for different adducts. By understanding the mol. dose and biol. of each adduct and comparing these to the dose-response for tumor induction, it may be possible to identify the most appropriate biomarkers for risk assessment. Recently, endogenous DNA adducts identical to those induced by known human carcinogens have been identified. These endogenously formed adducts may play an important role in human carcinogenesis.188Swenberg, J. A.; Bogdanffy, M. S.; Ham, A.; Holt, S.; Kim, A.; Morinello, E. J.; Ranasinghe, A.; Scheller, N.; Upton, P. B. Formation and repair of DNA adducts in vinyl chloride- and vinyl fluoride-induced carcinogenesis. IARC Sci. Publ. 1999, 29– 43[PubMed], [CAS], Google Scholar188https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXitF2qsbg%253D&md5=37d154fe244e303130c8d0da484d73c5Formation and repair of DNA adducts in vinyl chloride- and vinyl fluoride-induced carcinogenesisSwenberg, J. A.; Bogdanffy, M. S.; Ham, A.; Holt, S.; Kim, A.; Morinello, E. J.; Ranasinghe, A.; Scheller, N.; Upton, P. B.IARC Scientific Publications (1999), 150 (Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis), 29-43CODEN: IARCCD; ISSN:0300-5038. (International Agency for Research on Cancer)A review and discussion with many refs. on the formation and repair of DNA adducts assocd. with vinyl chloride and vinyl fluoride in exposed and control rodents and unexposed humans. Vinyl chloride is a known human and animal carcinogen that induces angiosarcomas of the liver. These vinyl halides induce etheno (ε) adducts that are identical to those formed after lipid peroxidn. Of these adducts, N2,3-ethenoguanine (εG) is present in the greatest amts. in tissues of exposed animals. After exposure to vinyl chloride for 4 wk, εG levels attain steady-state concns., such that the amt. of newly formed adducts equals the no. of adducts that are lost each day. The authors report the first dosimetry of εG in rats exposed to 0, 10, 100, or 1100 ppm vinyl chloride for 5 days or 4 wk. The no. of adducts increased in a supralinear manner. Exposure to 10 ppm vinyl chloride for 5 days caused a 2- to 3-fold increase in εG over that of the controls while a 4-wk exposure resulted in a 5-fold increase. This was confirmed with [13C2]vinyl chloride and by measuring exogenous and endogenous adducts in the same animals. Exposure to 100 ppm vinyl chloride for 4 wk caused a 25-fold increase in εG levels over that found in control rats while exposure to 1100 ppm resulted in a 42-fold increase. The amt. of endogenous εG was similar in liver DNA from rats and humans. A comparable response to exposure was seen in rats and mice exposed to 0, 25, 250, or 2500 ppm vinyl fluoride for 12 mo. There was a very high correlation between εG levels in rat and mouse liver at 12 mo and the incidence of hemangiosarcoma at 2 yr. The authors were able to demonstrate that the target cell population for angiosarcoma, the nonparenchymal cells, contained more εG than hepatocytes, even though nonparenchymal cells are exposed by diffusion of vinyl halide metabolites formed in hepatocytes. The expression of N-methylpurine-DNA glycosylase mRNA was induced in rat liver after exposure to either 25 or 2500 ppm vinyl fluoride. When this induction was investigated in hepatocytes and nonparenchymal cells, it was found that the latter had only 20% of the N-methylpurine-DNA glycosylase mRNA of hepatocytes, and that only the hepatocytes had induction of this expression after exposure to vinyl fluoride. Thus, the target cells for vinyl halide carcinogenesis have much lower expression of this DNA repair enzyme, which has been assocd. with etheno adduct repair.189Filser, J. G.; Jung, P.; Bolt, H. M. Increased acetone exhalation induced by metabolites of halogenated C1 and C2 compounds. Arch. Toxicol. 1982, 49, 107– 116, DOI: 10.1007/BF00332358[Crossref], [PubMed], [CAS], Google Scholar189https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XpvFSisA%253D%253D&md5=49db3c9a9010a263e6ead49675b5887fIncreased acetone exhalation induced by metabolites of halogenated C1 and C2 compoundsFilser, J. G.; Jung, P.; Bolt, H. M.Archives of Toxicology (1982), 49 (2), 107-16CODEN: ARTODN; ISSN:0340-5761.Rats were exposed, in a closed desiccator jar chamber, concns. of various halogenated C1 and C2 compds. at which the metabolizing capacities were satd. (Vmax conditions). Within the exposure period of 50 h concns. of the xenobiotic and of exhaled Me2CO [67-64-1] were monitored in the gas phase of the system. The quant. extent of Me2CO exhalation was dependent on the individual compd. examd. Me2CO exhalation was stimulated in presence of vinyl chloride [75-01-4], vinyl bromide [593-60-2], vinyl fluoride [75-02-5], vinylidene fluoride [75-38-7], cis- [156-59-2] and trans-1,2-dichloroethylene [156-60-5], trichloroethylene [79-01-6], perchloroethylene [127-18-4], CH2Cl2 [75-09-2], CHCl3 [67-66-3], CCl4 [56-23-5], and 1,1,2-trichloroethane [79-00-5]. No stimulation of Me2CO exhalation occurred with 1,1,1-trichloroethane [71-55-6] and with the ref. hydrocarbon n-hexane. Also, Me2CO exhalation was evoked by infusions of either fluoroacetate [144-49-0] or chloroacetate [14526-03-5], 2 anticipated or proven metabolites of some haloethylenes; the infusion rate of which were based on calcns. of the metabolic rates of vinylidene fluoride and of vinyl chloride, resp.190Ortiz de Montellano, P. R.; Kunze, K. L.; Beilan, H. S.; Wheeler, C. Destruction of cytochrome P-450 by vinyl fluoride, fluroxene, and acetylene. Evidence for a radical intermediate in olefin oxidation. Biochemistry 1982, 21, 1331– 1339, DOI: 10.1021/bi00535a035[ACS Full Text], [CAS], Google Scholar190https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XpvFGgtw%253D%253D&md5=c6623d20a258545ddd72e80cdde1750dDestruction of cytochrome P-450 by vinyl fluoride, fluroxene, and acetylene. Evidence for a radical intermediate in olefin oxidationOrtiz de Montellano, Paul R.; Kunze, Kent L.; Beilan, Hal S.; Wheeler, ConradBiochemistry (1982), 21 (6), 1331-9CODEN: BICHAW; ISSN:0006-2960.Vinyl fluoride, vinyl bromide, fluroxene (2,2,2-trifluoroethyl vinyl ether), and acetylene alkylate the prosthetic heme group of cytochrome P-450 enzymes which catalyze their metab. The alkylated heme moiety has been identified in all 4 cases, after carboxyl group methylation and demetalation, as the di-Me ester of N-(2-oxoethyl)protoporphyrin IX. The di-Me acetal deriv. of the aldehyde group in this structure was also isolated. The formation of the same prosthetic heme adduct with the 4 substrates requires introduction of an O at the trifluoroethoxy or halide-substituted terminus of the π bond and reaction of the unsubstituted terminus with a heme N atom. This reaction orientation is consistent with a radical intermediate, possibly formed by way of an initial π-bond radical cation, but is difficult to reconcile with a cationic intermediate. The occurrence of a radical intermediate in the oxidn. of olefins by cytochrome P-450 is thus suggested.191Odum, J.; Green, T. The metabolism and nephrotoxicity of tetrafluoroethylene in the rat. Toxicol. Appl. Pharmacol. 1984, 76, 306– 318, DOI: 10.1016/0041-008X(84)90012-7[Crossref], [PubMed], [CAS], Google Scholar191https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXptV2htw%253D%253D&md5=3d9f3ef28e97c4933a69a0bcaa5cc530The metabolism and nephrotoxicity of tetrafluoroethylene in the ratOdum, J.; Green, T.Toxicology and Applied Pharmacology (1984), 76 (2), 306-18CODEN: TXAPA9; ISSN:0041-008X.Exposure of rats to 6000 ppm tetrafluoroethylene (I) [116-14-3] for 6 h produced marked damage to the proximal tubule of the kidney with no effect on the liver. The toxicity was characterized by very high concns. of urinary glucose [50-99-7] and by marked increases in the concns. of several urinary enzymes. The no obsd. effect level for a 6-h exposure was 2000 ppm. I was metabolized to S-(1,1,2,2-tetrafluoroethyl)glutathione [94840-67-2] by rat liver fractions in vitro; the reaction was catalyzed by microsomal and cytosolic GSH S-transferase [50812-37-8]. The rate with microsomes was 4 times with cytosol fractions. Evidence for this metabolic pathway in vivo was obtained by the identification of the cysteinylglycine and cysteine conjugates of I in rat bile. Cytochrome P 450 [9035-51-2] oxidn., a common metabolic route for haloalkenes, does not appear to occur in the metab. of I. When administered orally to rats, the synthetic cysteine conjugate of I caused renal damage identical to that caused by I itself. The conjugate was metabolized by renal slices in vitro giving pyruvate, NH3, and a reactive species which caused marked inhibition of org. ion transport into slices. Purified renal βb-lyase [68652-57-3] also cleaved this conjugate giving stoichiometric amts. of pyruvate and ammonia. The nephrotoxicity of I may derive from the hepatic GSH conjugate of this compd. Following excretion and degrdn. of this conjugate in bile, the cysteine conjugate is reabsorbed and further metabolized in the kidney by the enzyme β-lyase to a cytotoxic species.192Keller, D. A.; Kennedy, G. L., Jr.; Ross, P. E.; Kelly, D. P.; Elliott, G. S. Toxicity of tetrafluoroethylene and S-(1,1,2, 2-tetrafluoroethyl)-l-cysteine in rats and mice. Toxicol. Sci. 2000, 56, 414– 423, DOI: 10.1093/toxsci/56.2.414[Crossref], [PubMed], [CAS], Google Scholar192https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXlsVCjur0%253D&md5=7d73e93c7116bae45ff2f939915cdaa5Toxicity of tetrafluoroethylene and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine in rats and miceKeller, Douglas A.; Kennedy, Gerald L., Jr.; Ross, Paul E.; Kelly, David P.; Elliott, Glenn S.Toxicological Sciences (2000), 56 (2), 414-423CODEN: TOSCF2; ISSN:1096-6080. (Oxford University Press)Groups of 25 female F344 rats and 25 female B6C3F1 mice were exposed to 0, 30, 300, 600, or 1200 ppm tetrafluoroethylene (TFE) by inhalation for up to 12 days. Another set of 25 female rats and 25 female mice of the same strains were given 0, 5, 20, or 50 mg/kg S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFE-CYS) by oral gavage for 12 days. Both 12-day exposure regimens consisted of exposures for 5 consecutive days, a weekend with no exposures, and 4 consecutive daily exposures following the weekend. Five animals per group were sacrificed after the first exposure, the fifth exposure, and the ninth exposure for evaluation of cell proliferation in the liver and kidney. The remaining animals in each group (up to 10) were sacrificed after the ninth exposure (test day 12) for pathol. evaluation of the liver, kidney, and spleen. Clin. pathol. evaluations were performed on test day 11 or 12. Inhalation of TFE by rats and mice caused slight microscopic changes in the kidneys of rats and mice but no histopathol. changes in the liver. In the kidney, administration of TFE-CYS by gavage caused severe microscopic changes in rats, moderate-to-severe changes in mice, and no microscopic changes in the liver. Cell proliferation was increased in the kidneys of rats and mice given TFE by inhalation and TFE-CYS by gavage. TFE-CYS also caused increased liver wts. and cell proliferation in the liver of rats and mice at the high doses. The cell proliferation response in the kidney and liver was transient in both species, being most pronounced after 5 days of exposure, and less evident or absent after 12 days of exposure. In the kidney, the cell proliferation and histopathol. response in rats was generally more pronounced than in mice. Kidney damage and cell proliferation were confined to the pars recta (P3) of the outer stripe of the outer medulla and medullary rays. Tubules in mice exposed to TFE and TFE-CYS had mostly regenerating cells by test day 12 while in rats the tubules still showed marked degeneration along with regeneration by the end of the study. The cortical labyrinth (P1 and P2 segments) was also affected at the 50-mg/kg dose of TFE-CYS in rats. Rats exposed to 50 mg/kg TFE-CYS had a mild anemia, and rats exposed to 1200 ppm TFE had slight, biol. inconsequential decreases in erythrocyte mass that may have been compd.-related. In spite of the rather pronounced histopathol. changes in the kidneys of rats exposed to TFE-CYS, there was no clin. chem. evidence for decreased kidney function. Increased levels of urinary fluoride were present in rats exposed to 300 ppm and greater of TFE and in rats exposed to 20 and 50 mg/kg TFE-CYS. The spleen was not affected in this study. Overall, the results of this study suggest that the effects of TFE could be attributed to the toxicity of TFE-CYS over the course of a 2-wk exposure, as all effects that were seen with TFE were also seen with TFE-CYS.193Puts, G. J.; Crouse, P.; Ameduri, B. M. Polytetrafluoroethylene: Synthesis and Characterization of the Original Extreme Polymer. Chem. Rev. 2019, 119, 1763– 1805, DOI: 10.1021/acs.chemrev.8b00458[ACS Full Text], [CAS], Google Scholar193https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVCltr0%253D&md5=5bb35db39b39c6c671a2e47c98627689Polytetrafluoroethylene: Synthesis and Characterization of the Original Extreme PolymerPuts, Gerard J.; Crouse, Philip; Ameduri, Bruno M.Chemical Reviews (Washington, DC, United States) (2019), 119 (3), 1763-1805CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. This review aims to be a comprehensive, authoritative, and crit. review of general interest to the chem. community (both academia and industry) as it contains an extensive overview of all published data on the homopolymn. of tetrafluoroethylene (TFE), detailing the TFE homopolymn. process and the resulting chem. and phys. properties. Several reviews and encyclopedia chapters on the properties and applications of fluoropolymers in general were published, including various reviews that extensively report copolymers of TFE (listed below). Despite this, a thorough review of the specific methods of synthesis of the homopolymer, and the relationships between synthesis conditions and the physicochem. properties of the material prepd., has not been available. This review intends to fill that gap. As known, PTFE and its marginally modified derivs. comprise some 60-65% of the total international fluoropolymer market with a global increase of ca. 7% per annum of its prodn. Numerous companies, such as Asahi Glass, Solvay Specialty Polymers, Daikin, DuPont/Chemours, Juhua, 3F and 3M/Dyneon, etc., produce TFE homopolymers. Such polymers, both high mol.-mass materials and waxes, are chem. inert, hydrophobic, and exhibit an excellent thermal stability and an exceptionally low coeff. of friction. These polymers find use in applications ranging from coatings and lubrication to pyrotechnics, and an extensive industry (electronic, aerospace, wires and cables, and textiles) was built around them. South Africa, being the third largest producer of fluorspar (CaF2), the precursor to hydrogen fluoride and fluorine, has embarked on an industrial initiative to locally beneficiate its fluorspar reserves, with the local prodn. of fluoropolymers being one projected outcome. As our manuscript focuses specifically on the homopolymn. of TFE (the starting point for all fluoropolymer industries), it will be of considerable use to start-up companies and other com. entities looking to enter the fluoropolymer market, and end-user companies. The manuscript commences with a short discussion on the synthesis and prodn. of TFE (both at industrial and lab. scales), including the safety aspects surrounding handling (since that monomer is regarded as explosive if brought into contact with oxygen due to the formation of peroxides), transport and storage, and then expands into detailed discussions dealing with aspects such as the various additives used (buffers, chain transfer agents, surfactants etc.), the solvent environment, and the reaction conditions. A further section reports the properties of PTFE with respect to the polymn. conditions and an overview on the specialized techniques used to characterize PTFE. Finally, the applications of PTFE into various topics, ranging from elec. insulation, tribol. to medical applications, and chem. resistant coatings and pyrotechnics are discussed.194NTP Technical Report on the Toxicology and Carcinogenesis Studies of Tetrafluoroethylene (CAS No. 116-14-3) in F344/N Rats and B6C3F1 Mice (Inhalation Studies). National Institutes of Health National Toxicology Program, April 1997. https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr450.pdf (accessed 2019-11-02).195Shim, J. Y.; Richard, A. M. Theoretical evaluation of two plausible routes for bioactivation of S-(1,1-difluoro-2,2-dihaloethyl)-l-cysteine conjugates: thiirane vs thionoacyl fluoride pathway. Chem. Res. Toxicol. 1997, 10, 103– 110, DOI: 10.1021/tx9600863[ACS Full Text], [CAS], Google Scholar195https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXotVWrtg%253D%253D&md5=672ddea96d18a414cc6cb5b869a8000dTheoretical Evaluation of Two Plausible Routes for Bioactivation of S-(1,1-Difluoro-2,2-dihaloethyl)-L-cysteine Conjugates: Thiirane vs. Thionoacyl Fluoride PathwayShim, Joong-Youn; Richard, Ann M.Chemical Research in Toxicology (1997), 10 (1), 103-110CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)Computational methods were applied to the investigation of two proposed metabolic pathways leading from the thiolate to either a thiirane or thionoacyl fluoride intermediate, both electrophilic species presumed capable of binding to proteins or DNA. Studied were six F-, Cl-, and Br-substituted 2,2-dihalo-1,1-difluoroethane-1-thiolates (2,2-dihalo-DFETs). Pathway preference was detd. for each thiolate by comparison of reaction energy profiles and activation energies. At all but the lowest level of ab initio theory, a thionoacyl fluoride pathway was predicted for 2,2-difluoro-DFET, while a thiirane pathway was energetically preferred for the brominated 2,2-dihalo-DFETs. These results offer a clear mechanism-based rationale for distinguishing 2,2-difluoro-DFET from the brominated 2,2-dihalo-DFETs, while the results are less clear for the 2,2-dichloro and 2-chloro-2-fluoro-DFETs, which at the highest level of ab initio treatment had a relatively small energy preference (2.4 kcal/mol) for the thiirane pathway. The predicted clear preference for a thiirane pathway for the brominated 2,2-dihalo-DFETs is not consistent with a recently proposed pathway involving α-thiolactone formation through a thionoacyl fluoride intermediate [Finkelstein, M. B., et al. (1995) J. Am. Chem. Soc. 117, 9590-9591], but is supported by results of a recent study providing exptl. evidence for thiirane formation from the brominated 2,2-dihalo-DFETs [Finkelstein, M. B., et al. (1996) Chem. Res. Toxicol. 9, 227-231].196Commandeur, J. N.; Brakenhoff, J. P.; De Kanter, F. J.; Vermeulen, N. P. Nephrotoxicity of mercapturic acids of three structurally related 2,2-difluoroethylenes in the rat. Indications for different bioactivation mechanisms. Biochem. Pharmacol. 1988, 37, 4495– 4504, DOI: 10.1016/0006-2952(88)90665-X[Crossref], [PubMed], [CAS], Google Scholar196https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXptVSltQ%253D%253D&md5=7d98aae03afd57c671238993632ebbd3Nephrotoxicity of mercapturic acids of three structurally related 2,2-difluoroethylenes in the rat. Indications for different bioactivation mechanismsCommandeur, J. N. M.; Brakenhoff, J. P. G.; De Kanter, F. J. J.; Vermeulen, N. P. E.Biochemical Pharmacology (1988), 37 (23), 4495-504CODEN: BCPCA6; ISSN:0006-2952.The biotransformation and the hepato- and nephrotoxicity of the mercapturic acids (N-acetyl-1-cysteine S-conjugates) of three structurally related 2,2-difluoroethylenes were investigated in vivo in the rat. All mercapturic acids appeared to cause nephrotoxicity, without any measurable effect on the liver. The mercapturic acid of tetrafluoroethylene (TFE-NAC) appeared to be the most potent nephrotoxin, causing toxicity upon an i.p. dose of 50 μmol/kg. The mercapturic acids of 1,1-dichloro-2,2-difluoroethylene (DCDFE-NAC) and 1,1-dibromo-2,2-difluoroethylene (DBDFE-NAC) were nephrotoxic at slightly higher doses, i.e. at 75 and 100 μmol/kg, resp. In the urine of TFE-NAC-treated rats significant amts. of difluoroacetic acid (DFAA) could be detected. With increasing doses, the relative amt. of DFAA in urine increased progressively (5-18% of dose). In urine of rats treated with DCDFE-NAC and DBDFE-NAC, however, the corresponding dihaloacetic acids, dichloroacetic acid, and dibromoacetic acid, could not be detected. Formation of DFAA and pyruvate could also be obsd. during in vitro metab. of the cysteine conjugate of tetrafluoroethylene (TFE-CYS) by rat renal cytosol. Inhibition by aminooxyacetic acid pointed to a β-lyase dependency for the DFAA-formation. Next to DFAA and pyruvate, also formation of hydrogen sulfide and thiosulfate could be detected. These results suggest that TFE-CYS is bioactivated to a significant extent to difluorothionacyl fluoride, which most likely is subsequently hydrolyzed to difluorothio(no)acetic acid and difluoroacetic acid. According to formation of pyruvate, the cysteine conjugates derived from DCDFE-NAC and DBDFE-NAC also were efficiently metabolized by rat renal β-lyase. However, the formation of corresponding dihaloacetic acids, dichloroacetic acid, and dibromoacetic acid, could not be detected in vitro at all. Only very small amts. of hydrogen sulfide and thiosulfate were detected. Evidently bioactivation of the latter two conjugates to a dichloro- or dibromothionoacyl fluoride represents only a minor route. Because of better leaving group abilities of chloride and bromide compared to fluoride, rearrangement of the initially formed ethanethiol to a thiirane might be favored. Based on the present in vivo and in vitro data, it is concluded that the nephrotoxicity of the structurally related mercapturic acids of 2,2-difluoroethylenes is dependent on halogen substitution and presumably the result of at least two different mechanisms of bioactivation.197Potter, C. L.; Gandolfi, A. J.; Nagle, R.; Clayton, J. W. Effects of inhaled chlorotrifluoroethylene and hexafluoropropene on the rat kidney. Toxicol. Appl. Pharmacol. 1981, 59, 431– 440, DOI: 10.1016/0041-008X(81)90295-7[Crossref], [PubMed], [CAS], Google Scholar197https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXkvFWqsLw%253D&md5=5a70e9211a2487aa7a174fae8bd1fc1eEffects of inhaled chlorotrifluoroethylene and hexafluoropropene on the rat kidneyPotter, C. L.; Gandolfi, A. J.; Nagle, R.; Clayton, J. W.Toxicology and Applied Pharmacology (1981), 59 (3), 431-40CODEN: TXAPA9; ISSN:0041-008X.Male rats were subjected to a 4.0-h inhalation exposure to chlorotrifluoroethylene (CTFE) [79-38-9] (100-540 ppm) or hexafluoropropene (HFP) [116-15-4] (380-1200 ppm). Within 2 days following exposure, the rats exhibited dose-related proximal tubular necrosis, diuresis, increases in urinary fluoride, urinary lactic dehydrogenase (LDH) [9001-60-9] activity, serum creatinine [60-27-5], and BUN. The toxicities of CTFE and HFP were similar except that CTFE was the more potent renal toxin and HFP produced necrosis of the pars recta and pars convoluta portions of the proximal tubule, while CTFE produced necrosis of only the pars recta. At the lowest exposure concns., diuresis was the most sensitive index of toxicity manifesting 50% increases in water intake and 25% decreases in urine osmolality. Increases in urinary LDH activity correlated with the degree of proximal renal tubular necrosis, with ≥100-fold increases at the highest concns. of CTFE and HFP. At 100 ppm, CTFE induced renal dysfunction (mild diuresis), but no significant increase in urinary LDH nor necrosis was apparent. All concns. of HFP studied produced necrosis within 24 h postexposure with tubular cell regeneration apparent within 4 days.198Commandeur, J. N.; Oostendorp, R. A.; Schoofs, P. R.; Xu, B.; Vermeulen, N. P. Nephrotoxicity and hepatotoxicity of 1,1-dichloro-2,2-difluoroethylene in the rat. Indications for differential mechanisms of bioactivation. Biochem. Pharmacol. 1987, 36, 4229– 4237, DOI: 10.1016/0006-2952(87)90663-0[Crossref], [PubMed], [CAS], Google Scholar198https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXntVaqtg%253D%253D&md5=d01930644460f08f06c66493ef9f4145Nephrotoxicity and hepatotoxicity of 1,1-dichloro-2,2-difluoroethylene in the rat. Indications for differential mechanisms of bioactivationCommandeur, J. N. M.; Oostendorp, R. A. J.; Schoofs, P. R.; Xu, B.; Vermeulen, N. P. E.Biochemical Pharmacology (1987), 36 (24), 4229-37CODEN: BCPCA6; ISSN:0006-2952.1,1-Dichloro-2,2-difluoroethylene (DCDFE) produced marked nephrotoxicity in rats after an i.p. dose of 150 μmol/kg. At doses higher than 375 μmol/kg, DCDFE also produced hepatotoxicity. Aminooxyacetic acid, an inhibitor of cysteine conjugate β-lyase, appeared to be slightly nephrotoxic in rats. Nevertheless it exerted an inhibitory effect on the nephrotoxicity of DCDFE. The N-acetylcysteine conjugate of DCDFE was identified as a major urinary metabolite of DCDFE. When administered as such, this conjugate appeared to be a potent nephrotoxin, without any effect on the liver, indicating that glutathione conjugation of DCDFE is most likely a bioactivation step for nephrotoxicity. The appearance of traces of chlorodifluoroacetic acid in urine of rats treated with higher doses of DCDFE indicates the existence of an oxidative pathway of metab. of DCDFE, probably involving epoxidn. by hepatic mixed-function oxidases. It is speculated that the latter route might account for the hepatotoxicity at higher doses of DCDFE. The nephro- and hepatotoxicity of DCDFE, therefore, most likely are the result of 2 different mechanisms of bioactivation.199Ishmael, J.; Lock, E. A. Nephrotoxicity of hexachlorobutadiene and its glutathione-derived conjugates. Toxicol. Pathol. 1986, 14, 258– 262, DOI: 10.1177/019262338601400216[Crossref], [PubMed], [CAS], Google Scholar199https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXitVyqtrg%253D&md5=5cabde1e399a4d55ab3ba70e1c99bbbaNephrotoxicity of hexachlorobutadiene and its glutathione-derived conjugatesIshmael, John; Lock, Edward A.Toxicologic Pathology (1986), 14 (2), 258-62CODEN: TOPADD; ISSN:0192-6233.The nephrotoxicity of hexachlor-1,3-butadiene (HCBD) [87-68-3], its GSH conjugate (HCBD-GSH) [89021-88-5], cysteine conjugate (HCBD-CYS) [87619-82-7], and its N-acetylcysteine conjugate (HCBD-NAC) [89784-39-4] were compared in male and female Alderley Park rats. Rats, 6-8 wk of age, were given a single i.p. injection of HCBD or its conjugates and killed 24 h later. Nephrotoxicity was assessed by histol. examn. and plasma urea. All 3 conjugates produced an elevation of plasma urea and proximal renal tubular necrosis with a similar localization in the pars recta as seen with HCBD. All the conjugates were more nephrotoxic than HCBD itself. HCBD was ∼4 times more toxic to female rats than to males. This sex difference is also shown by all the HCBD metabolites.200Tysoe, C.; Withers, S. G. Fluorinated mechanism-based inhibitors: common themes and recent developments. Curr. Top. Med. Chem. 2014, 14, 865– 874, DOI: 10.2174/1568026614666140202204602[Crossref], [PubMed], [CAS], Google Scholar200https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXlt1Srsbs%253D&md5=033531feb3cb8aaae337fa4ade61af3bFluorinated Mechanism-Based Inhibitors: Common Themes and Recent DevelopmentsTysoe, Christina; Withers, Stephen G.Current Topics in Medicinal Chemistry (Sharjah, United Arab Emirates) (2014), 14 (7), 865-874CODEN: CTMCCL; ISSN:1568-0266. (Bentham Science Publishers Ltd.)A review. Mechanism-based inhibitors are relatively chem. inert compds. that become activated when processed by their target enzyme, leading to covalent enzyme inactivation. Fluorine substitution confers a no. of properties that are beneficial to the chem. of such inhibitors and to their potential use as pharmaceuticals, and indeed several fluorinated mechanism-based inhibitors have made it to clin. usage over the past 50 years. Well-known examples are the 5- fluorouracil metabolite, 5-fluoro-2'-deoxyuridine-5'-monophosphate, which is used in the treatment of cancer, and α - difluoromethylornithine for the treatment of African sleeping sickness. As the prevalence of fluorine in medicinal chem. continues to rise, more and more medically relevant fluorinated mechanism-based inhibitors are being developed with a variety of interesting properties and uses.201Belter, A.; Skupinska, M.; Giel-Pietraszuk, M.; Grabarkiewicz, T.; Rychlewski, L.; Barciszewski, J. Squalene monooxygenase - a target for hypercholesterolemic therapy. Biol. Chem. 2011, 392, 1053– 1075, DOI: 10.1515/BC.2011.195[Crossref], [PubMed], [CAS], Google Scholar201https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XivFSrtLk%253D&md5=9d4af8d79ed6ed0797c74c6b53a42ddbSqualene monooxygenase - a target for hypercholesterolemic therapyBelter, Agnieszka; Skupinska, Miroslawa; Giel-Pietraszuk, Malgorzata; Grabarkiewicz, Tomasz; Rychlewski, Leszek; Barciszewski, JanBiological Chemistry (2011), 392 (12), 1053-1075CODEN: BICHF3; ISSN:1431-6730. (Walter de Gruyter GmbH & Co. KG)A review. Squalene monooxygenase catalyzes the epoxidn. of C-C double bond of squalene to yield 2,3-oxidosqualene, the key step of sterol biosynthesis pathways in eukaryotes. Sterols are essential compds. of these organisms and squalene epoxidn. is an important regulatory point in their synthesis. Squalene monooxygenase downregulation in vertebrates and fungi decreases synthesis of cholesterol and ergosterol, resp., which makes squalene monooxygenase a potent and attractive target of hypercholesterolemia and antifungal therapies. Currently some fungal squalene monooxygenase inhibitors (terbinafine, naftifine, butenafine) are in clin. use, whereas mammalian enzymes' inhibitors are still under investigation. Research on new squalene monooxygenase inhibitors is important due to the prevalence of hypercholesterolemia and the lack of both sufficient and safe remedies. In this paper we (i) review data on activity and the structure of squalene monooxygenase, (ii) present its inhibitors, (iii) compare current strategies of lowering cholesterol level in blood with some of the most promising strategies, (iv) underline advantages of squalene monooxygenase as a target for hypercholesterolemia therapy, and (v) discuss safety concerns about hypercholesterolemia therapy based on inhibition of cellular cholesterol biosynthesis and potential usage of squalene monooxygenase inhibitors in clin. practice. After many years of use of statins there is some clin. evidence for their adverse effects and only partial effectiveness. Currently they are drugs of choice but are used with many restrictions, esp. in case of children, elderly patients and women of childbearing potential. Certainly, for the next few years, statins will continue to be a suitable tool for cost-effective cardiovascular prevention; however research on new hypolipidemic drugs is highly desirable. We suggest that squalene monooxygenase inhibitors could become the hypocholesterolemic agents of the future.202Cirmena, G.; Franceschelli, P.; Isnaldi, E.; Ferrando, L.; De Mariano, M.; Ballestrero, A.; Zoppoli, G. Squalene epoxidase as a promising metabolic target in cancer treatment. Cancer Lett. 2018, 425, 13– 20, DOI: 10.1016/j.canlet.2018.03.034[Crossref], [PubMed], [CAS], Google Scholar202https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntVOit70%253D&md5=43d5d59d5d9fd3b035826480232ee302Squalene epoxidase as a promising metabolic target in cancer treatmentCirmena, Gabriella; Franceschelli, Paola; Isnaldi, Edoardo; Ferrando, Lorenzo; De Mariano, Marilena; Ballestrero, Alberto; Zoppoli, GabrieleCancer Letters (New York, NY, United States) (2018), 425 (), 13-20CODEN: CALEDQ; ISSN:0304-3835. (Elsevier)A review. Oncogenic alteration of the cholesterol synthesis pathway is a recognized mechanism of metabolic adaptation. In the present review, we focus on squalene epoxidase (SE), one of the two rate-limiting enzymes in cholesterol synthesis, retracing its history since its discovery as an antimycotic target to its description as an emerging metabolic oncogene by amplification with clin. relevance in cancer. We review the published literature assessing the assocn. between SE over-expression and poor prognosis in this disease. We assess the works demonstrating how SE promotes tumor cell proliferation and migration, and displaying evidence of cancer cell demise in presence of human SE inhibitors in in vitro and in vivo models. Taken together, robust scientific evidence has by now accumulated pointing out SE as a promising novel therapeutic target in cancer treatment.203Ono, T.; Nakazono, K.; Kosaka, H. Purification and partial characterization of squalene epoxidase from rat liver microsomes. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1982, 709, 84– 90, DOI: 10.1016/0167-4838(82)90424-1[Crossref], [PubMed], [CAS], Google Scholar203https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3sXhtlGhtg%253D%253D&md5=7ed26117db6e34c54a37e21eebb86e3bPurification and partial characterization of squalene epoxidase from rat liver microsomesOno, Teruo; Nakazono, Kiyoshi; Kosaka, HirokoBiochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (1982), 709 (1), 84-90CODEN: BBAEDZ; ISSN:0167-4838.Squalene epoxidase (EC 1.14.99.7) (I) was purified to apparent homogeneity from rat liver microsomes. The purifn. was carried out by solubilization of microsomes by Triton X-100, fractionation with ion-exchangers, hydroxyapatite, Cibacron Blue Sepharose 4B, and chromatofocusing column chromatog. A total purifn. of 143-fold over the 1st DEAE-cellulose fraction was achieved. Purified I gave a single major band on SDS-polyacrylamide gel electrophoresis and the mol. wt. was estd. to be 51,000 as a single polypeptide chain. I showed no distinct absorption spectrum in the visible regions. The I activity was reconstituted with the purified enzyme, NADPH-cytochrome P 450 reductase, FAD, NADPH, and O2 in the presence of Triton X-100. The apparent Km for squalene and FAD were 13 and 5 μM, resp. The Vmax was ∼186 nmol/mg protein/30 min for 2,3-oxidosqualene. I activity was not inhibited by potent inhibitors of cytochrome P 450. Apparently, I is distinct from cytochrome P 450 isoenzymes.204Moore, W. R.; Schatzman, G. L.; Jarvi, E. T.; Gross, R. S.; McCarthy, J. R. Terminal difluoro olefin analogs of squalene are time-dependent inhibitors of squalene epoxidase. J. Am. Chem. Soc. 1992, 114, 360– 361, DOI: 10.1021/ja00027a056[ACS Full Text], [CAS], Google Scholar204https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xjs1Oqtg%253D%253D&md5=00d486607e27a6cce124e6925eacf244Terminal difluoro olefin analogs of squalene are time-dependent inhibitors of squalene epoxidaseMoore, William R.; Schatzman, Gerald L.; Jarvi, Esa T.; Gross, Raymond S.; McCarthy, James R.Journal of the American Chemical Society (1992), 114 (1), 360-1CODEN: JACSAT; ISSN:0002-7863.Squalene epoxidase is an essential enzyme in the biosynthesis of cholesterol in humans and ergosterol in fungi and thus represents a potential target for hypocholesterolemic and antifungal therapeutics. The design, synthesis, and enzyme inhibition data for a series of mono- and difluoro olefin squalene analogs, such as (E,E,E,E)-1,1-difluoro-5,9,14,18,22-pentamethyl-1,5,9,13,17,21-tricosahexaene (I), are reported. Difluoro olefin squalene analogs I and II were time-dependent inhibitors of squalene epoxidase from rat liver; Ki and kinact values were 4 μM and 0.16 min-1 and 8 μM and 0.12 min-1 for I and II, resp. Difluoro olefin compds. with subtle structural changes from I had substantially reduced potency: a homolog of I increased in chain length and a C-2 Me-substituted I were not time-dependent inhibitors and had IC50 values of >100 μM. The inhibition of squalene epoxidase by difluoro olefins I and II appeared to be enzyme-activated. Monofluoro olefin squalene analogs were reversible inhibitors of squalene epoxidase from rat liver with IC50 values of 100 and 47 μM, resp. A new method for the synthesis of 1-(alkyl)-1 fluoro olefins is presented.205Wolfe, M. S.; Borchardt, R. T. S-adenosyl-l-homocysteine hydrolase as a target for antiviral chemotherapy. J. Med. Chem. 1991, 34, 1521– 1530, DOI: 10.1021/jm00109a001[ACS Full Text], [CAS], Google Scholar205https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXit1Smt70%253D&md5=445e649eb2925474f4b7abade9ab0cb6S-Adenosyl-L-homocysteine hydrolase as a target for antiviral chemotherapyWolfe, Michael S.; Borchardt, Ronald T.Journal of Medicinal Chemistry (1991), 34 (5), 1521-30CODEN: JMCMAR; ISSN:0022-2623.A review with 117 refs. on the antiviral S-adenosylhomocysteine hydrolase inhibitors with discussions on their cytotoxicity and present and future prospects as antiviral drugs as well as tools for studying the physiol. function of the hydrolase.206Reddy, M. C.; Kuppan, G.; Shetty, N. D.; Owen, J. L.; Ioerger, T. R.; Sacchettini, J. C. Crystal structures of Mycobacterium tuberculosis S-adenosyl-l-homocysteine hydrolase in ternary complex with substrate and inhibitors. Protein Sci. 2008, 17, 2134– 2144, DOI: 10.1110/ps.038125.108[Crossref], [PubMed], [CAS], Google Scholar206https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVKgt7vI&md5=c512b956d68fc1db9fcfc2af88fd259aCrystal structures of Mycobacterium tuberculosis S-adenosyl-L-homocysteine hydrolase in ternary complex with substrate and inhibitorsReddy, Manchi C. M.; Kuppan, Gokulan; Shetty, Nishant D.; Owen, Joshua L.; Ioerger, Thomas R.; Sacchettini, James C.Protein Science (2008), 17 (12), 2134-2144CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)S-adenosylhomocysteine hydrolase (SAHH) is a ubiquitous enzyme that plays a central role in methylation-based processes by maintaining the intracellular balance between S-adenosylhomocysteine (SAH) and S-adenosylmethionine. We report the first prokaryotic crystal structure of SAHH, from Mycobacterium tuberculosis (Mtb), in complex with adenosine (ADO) and NAD. Structures of complexes with three inhibitors are also reported: 3'-keto aristeromycin (ARI), 2-fluoroadenosine, and 3-deazaadenosine. The ARI complex is the first reported structure of SAHH complexed with this inhibitor, and confirms the oxidn. of the 3' hydroxyl to a planar keto group, consistent with its prediction as a mechanism-based inhibitor. We demonstrate the in vivo enzyme inhibition activity of the three inhibitors and also show that 2-fluoroadenosine has bactericidal activity. While most of the residues lining the ADO-binding pocket are identical between Mtb and human SAHH, less is known about the binding mode of the homocysteine (HCY) appendage of the full substrate. We report the 2.0 Å resoln. structure of the complex of SAHH cocrystd. with SAH. The most striking change in the structure is that binding of HCY forces a rotation of His-363 around the backbone to flip out of contact with the 5' hydroxyl of the ADO and opens access to a nearby channel that leads to the surface. This complex suggests that His-363 acts as a switch that opens up to permit binding of substrate, then closes down after release of the cleaved HCY. Differences in the entrance to this access channel between human and Mtb SAHH are identified.207Mehdi, S.; Jarvi, E. T.; Koehl, J. R.; McCarthy, J. R.; Bey, P. The mechanism of inhibition of S-adenosyl-l-homocysteine hydrolase by fluorine-containing adenosine analogs. J. Enzyme Inhib. 1990, 4, 1– 13, DOI: 10.3109/14756369009030383[Crossref], [PubMed], [CAS], Google Scholar207https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXhslWhtg%253D%253D&md5=8855633bcc7b81de34f12490b124bcd3The mechanism of inhibition of S-adenosyl-L-homocysteine hydrolase by fluorine-containing adenosine analogsMehdi, Shujaath; Jarvi, Esa T.; Koehl, Jack R.; McCarthy, James R.; Bey, PhilippeJournal of Enzyme Inhibition (1990), 4 (1), 1-13CODEN: ENINEG; ISSN:8755-5093.(Z)-4',5'-Didehydro-5'-deoxy-5'-fluoroadenosine (I), 5'-deoxy-5'-difluoroadenosine (II), and 4',5'-didehydro-5'-deoxy-5'-fluoroarabinosyl-adenosine (III) are inhibitors of rat liver S-adenosyl-L-homocysteine hydrolase. I and II are time-dependent and irreversible inhibitors of the enzyme. Both I and II are oxidized by enzyme-bound NAD to produce NADH, and fluoride anion is formed in the inactivation reaction (0.7 to 1.0 mol fluoride/mol of enzyme subunit, and 1.7 mol fluoride/mol of enzyme subunit from I and II, resp.). The enzyme is stoichiometrically labeled with [8-3H]-I, but the label is lost upon denaturation of the protein either with or without treatment of the labeled complex with sodium borohydride. III, the arabino deriv. of I, is a competitive inhibitor of the enzyme. The mechanism of the inhibition of S-adenosyl-L-homocysteine hydrolase by these inhibitors is discussed.208Lee, K. M.; Choi, W. J.; Lee, Y.; Lee, H. J.; Zhao, L. X.; Lee, H. W.; Park, J. G.; Kim, H. O.; Hwang, K. Y.; Heo, Y. S.; Choi, S.; Jeong, L. S. X-ray crystal structure and binding mode analysis of human S-adenosylhomocysteine hydrolase complexed with novel mechanism-based inhibitors, haloneplanocin A analogues. J. Med. Chem. 2011, 54, 930– 938, DOI: 10.1021/jm1010836[ACS Full Text], [CAS], Google Scholar208https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXlt1Ortw%253D%253D&md5=667394f7f2c22c2aed24f9a4a84bc7a9X-ray Crystal Structure and Binding Mode Analysis of Human S-Adenosylhomocysteine Hydrolase Complexed with Novel Mechanism-Based Inhibitors, Haloneplanocin A AnaloguesLee, Kang Man; Choi, Won Jun; Lee, Yoonji; Lee, Hyun Joo; Zhao, Long Xuan; Lee, Hyuk Woo; Park, Jae Gyu; Kim, Hea Ok; Hwang, Kwang Yeon; Heo, Yong-Seok; Choi, Sun; Jeong, Lak ShinJournal of Medicinal Chemistry (2011), 54 (4), 930-938CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The x-ray crystal structure of human S-adenosylhomocysteine (AdoHcy) hydrolase was first detd. as a tetrameric form bound with the novel mechanism-based inhibitor fluoroneplanocin A (4b). The crystd. enzyme complex showed the closed conformation and turned out to be the intermediate of mechanism-based inhibition. It confirmed that the cofactor depletion by 3'-oxidn. of fluoroneplanocin A contributes to the enzyme inhibition along with the irreversible covalent modification of AdoHcy hydrolase. In addn., a series of haloneplanocin A analogs (4b-e and 5b-e) were designed and synthesized to characterize the binding role and reactivity of the halogen substituents and the 4'-CH2OH group. The biol. evaluation and mol. modeling studies identified the key pharmacophores and structural requirements for the inhibitor binding of AdoHcy hydrolase. The inhibitory activity was decreased as the size of the halogen atom increased and/or if the 4'-CH2OH group was absent. These results could be utilized to design new therapeutic agents operating via AdoHcy hydrolase inhibition.209Jeong, L. S.; Yoo, S. J.; Lee, K. M.; Koo, M. J.; Choi, W. J.; Kim, H. O.; Moon, H. R.; Lee, M. Y.; Park, J. G.; Lee, S. K.; Chun, M. W. Design, synthesis, and biological evaluation of fluoroneplanocin A as the novel mechanism-based inhibitor of S-adenosylhomocysteine hydrolase. J. Med. Chem. 2003, 46, 201– 203, DOI: 10.1021/jm025557z[ACS Full Text], [CAS], Google Scholar209https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XpsVegtbo%253D&md5=1d68e769c44b8cf937fb1ed50aba7acfDesign, Synthesis, and Biological Evaluation of Fluoroneplanocin A as the Novel Mechanism-Based Inhibitor of S-Adenosylhomocysteine HydrolaseJeong, Lak Shin; Yoo, Su Jeong; Lee, Kang Man; Koo, Mi Jeong; Choi, Won Jun; Kim, Hea Ok; Moon, Hyung Ryong; Lee, Min Young; Park, Jae Gyu; Lee, Sang Kook; Chun, Moon WooJournal of Medicinal Chemistry (2003), 46 (2), 201-203CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Fluoroneplanocin A was designed as a novel mechanism-based inhibitor of S-adenosylhomocysteine hydrolase (SAH) and efficiently synthesized via an electrophilic vinyl fluorination reaction (n-BuLi, N-fluorobenzenesulfonimide at -78 °C). Fluoroneplanocin A exhibited 2-fold more potent SAH inhibitory activity than the parent neplanocin A. A new mechanism of irreversible inhibition discovered in this work might provide new alternatives in the design of a different class of antiviral agents operating via SAH inhibition.210Rosen, T. C.; Yoshida, S.; Frohlich, R.; Kirk, K. L.; Haufe, G. Fluorinated phenylcyclopropylamines. 2. Effects of aromatic ring substitution and of absolute configuration on inhibition of microbial tyramine oxidase. J. Med. Chem. 2004, 47, 5860– 5871, DOI: 10.1021/jm049957t[ACS Full Text], [CAS], Google Scholar210https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXoslSgtr4%253D&md5=f997059cce8757712c33518b96a56677Fluorinated Phenylcyclopropylamines. 2. Effects of Aromatic Ring Substitution and of Absolute Configuration on Inhibition of Microbial Tyramine OxidaseRosen, Thomas C.; Yoshida, Shinichi; Froehlich, Roland; Kirk, Kenneth L.; Haufe, GuenterJournal of Medicinal Chemistry (2004), 47 (24), 5860-5871CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of para-substituted diastereopure cis- and trans-2-fluoro-2-arylcyclopropylamines were synthesized and these were investigated as inhibitors of microbial tyramine oxidase from Arthrobacter sp. All compds. were shown to be competitive inhibitors of this enzyme. The nature of the para-substituents in the more potent trans-isomer (cis-relationship between fluorine and the amino group) of 2-fluoro-2-arylcyclopropylamine influenced the inhibitory potency in a consistent fashion. Thus, electron-withdrawing groups (F, Cl) slightly decreased the activity, while the Me group (+ I substituent) increased the activity by a factor of ∼ 7 compared to trans-2-fluoro-2-phenylcyclopropylamine and by a factor of 90 compared to tranylcypromine. Activity also was strongly dependent on the abs. configuration. The (1S,2S)-enantiomer of 2-fluoro-2-phenylcyclopropylamine was an excellent inhibitor of tyramine oxidase whereas the (1R,2R)-enantiomer was essentially devoid of activity.211McDonald, I. A.; Lacoste, J. M.; Bey, P.; Palfreyman, M. G.; Zreika, M. Enzyme-activated irreversible inhibitors of monoamine oxidase: phenylallylamine structure-activity relationships. J. Med. Chem. 1985, 28, 186– 193, DOI: 10.1021/jm00380a007[ACS Full Text], [CAS], Google Scholar211https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXlvValsg%253D%253D&md5=c7f4e34858042219fd1395e6e144a086Enzyme-activated irreversible inhibitors of monoamine oxidase: phenylallylamine structure-activity relationshipsMcDonald, Ian A.; Lacoste, Jean Michel; Bey, Philippe; Palfreyman, Michael G.; Zreika, MoniqueJournal of Medicinal Chemistry (1985), 28 (2), 186-93CODEN: JMCMAR; ISSN:0022-2623.The title compds. I (R and R1 = H, Br, Cl or F; R2 = H or OMe; R3 = H, OH, OMe or CF3; R4 = H, Cl, OH, Me, or OMe) were prepd. as HCl salts by several routes and evaluated in vitro by incubation with a crude rat brain mitochondrial monoamine oxidase (MAO) prepn., and in vivo in mice. With the exception of (Z)-3-chloro-2-phenylallylamine, which acted as a competitive inhibitor, all I caused a time-dependent inhibition of MAO when selective substrates were used. (E)-3-Fluoro-2-(4-methoxyphenyl)- and 2-(3,4-dimethoxyphenyl)-3-fluoroallylamine were as selective for the B form of MAO as deprenyl. (E)-3-Fluoro-2-phenylallylamine given to mice at 1 mg/kg, i.p., gave good inhibition of MAO in the brain and heart which lasted ≤48 h. Structure-activity relations were discussed.212Bortolato, M.; Chen, K.; Shih, J. C. Monoamine oxidase inactivation: from pathophysiology to therapeutics. Adv. Drug Delivery Rev. 2008, 60, 1527– 1533, DOI: 10.1016/j.addr.2008.06.002[Crossref], [PubMed], [CAS], Google Scholar212https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtFWltbbK&md5=9085ccfdebab364e9ea78995bcfc16d1Monoamine oxidase inactivation: From pathophysiology to therapeuticsBortolato, Marco; Chen, Kevin; Shih, Jean C.Advanced Drug Delivery Reviews (2008), 60 (13-14), 1527-1533CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)A review. Monoamine oxidases (MAOs) A and B are mitochondrial bound isoenzymes which catalyze the oxidative deamination of dietary amines and monoamine neurotransmitters, such as serotonin, norepinephrine, dopamine, β-phenylethylamine and other trace amines. The rapid degrdn. of these mols. ensures the proper functioning of synaptic neurotransmission and is critically important for the regulation of emotional behaviors and other brain functions. The byproducts of MAO-mediated reactions include several chem. species with neurotoxic potential, such as hydrogen peroxide, ammonia and aldehydes. As a consequence, it is widely speculated that prolonged excessive activity of these enzymes may be conducive to mitochondrial damages and neurodegenerative disturbances. In keeping with these premises, the development of MAO inhibitors has led to important breakthroughs in the therapy of several neuropsychiatric disorders, ranging from mood disorders to Parkinson's disease. Furthermore, the characterization of MAO knockout (KO) mice has revealed that the inactivation of this enzyme produces a no. of functional and behavioral alterations, some of which may be harnessed for therapeutic aims. In this article, we discuss the intriguing hypothesis that the attenuation of the oxidative stress induced by the inactivation of either MAO isoform may contribute to both antidepressant and antiparkinsonian actions of MAO inhibitors. This possibility further highlights MAO inactivation as a rich source of novel avenues in the treatment of mental disorders.213Laine, K.; Anttila, M.; Helminen, A.; Karnani, H.; Huupponen, R. Dose linearity study of selegiline pharmacokinetics after oral administration: evidence for strong drug interaction with female sex steroids. Br. J. Clin. Pharmacol. 1999, 47, 249– 254, DOI: 10.1046/j.1365-2125.1999.00891.x[Crossref], [PubMed], [CAS], Google Scholar213https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXisVKiu7w%253D&md5=b47fecbe9ff7e5659e5b1659370ff5f8Dose linearity study of selegiline pharmacokinetics after oral administration: evidence for strong drug interaction with female sex steroidsLaine, Kari; Anttila, Markku; Helminen, Antti; Karnani, Hari; Huupponen, RistoBritish Journal of Clinical Pharmacology (1999), 47 (3), 249-254CODEN: BCPHBM; ISSN:0306-5251. (Blackwell Science Ltd.)The purpose of this study was to characterize the dose relation of selegiline and desmethylselegiline pharmacokinetics within the selegiline dose range from 5 to 40 mg. Eight female subjects, of whom four were using oral contraceptives, ingested a single dose of 5 mg, 10 mg, 20 mg or 40 mg of selegiline HCl in an open four-period randomized study. Concns. of selegiline and desmethylselegiline in serum were measured by gas chromatog. for 5 h. As it became evident that the use of oral steroids had a drastic effect on selegiline concns., the pharmacokinetic analyses were performed sep. for oral contraceptive users and those not receiving any concomitant medication. The total AUC and Cmax of selegiline were 10-to 20-fold higher in those subjects taking oral steroids compared with subjects with no concomitant medication; this finding was consistent and statistically significant at all the four dose levels. The dose linearity of selegiline pharmacokinetics failed to be demonstrated in both groups. The AUC and Cmax of desmethylselegiline were only moderately higher (about 1.5-fold; P=NS at each dose level) in the subjects taking oral steroids than in those not receiving concomitant medication. The AUC values of desmethylselegiline increased in a dose linear manner in subjects with no concomitant medication, but not in the oral steroid group. The metabolic ratio (AUC(desmethylselegiline)/AUC(selegiline)) was several-fold lower in the group receiving oral steroids compared with the no-concomitant-medication group (at all the four dose levels). Concomitant use of oral contraceptives caused a drastic (20-fold) increase in the oral bioavailability of selegiline. The highly significant difference in the metabolic ratio between the groups provides evidence that the mechanism of the interaction between selegiline and female sex steroids involves reduced N-demethylation of selegiline. The present results suggest that concomitant use of selegiline with exogenous female sex steroids should be avoided or the dosage of selegiline should be reduced to minimize the risks of selegiline related adverse drug reactions.214Zreika, M.; Fozard, J. R.; Dudley, M. W.; Bey, P.; McDonald, I. A.; Palfreyman, M. G. MDL 72,974: a potent and selective enzyme-activated irreversible inhibitor of monoamine oxidase type B with potential for use in Parkinson’s disease. J. Neural Transm.: Parkinson's Dis. Dementia Sect. 1989, 1, 243– 254, DOI: 10.1007/BF02263478[Crossref], [PubMed], [CAS], Google Scholar214https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK3c%252FovVOnsg%253D%253D&md5=524fad71ec17bc2a9e6baadd825b07deMDL 72,974: a potent and selective enzyme-activated irreversible inhibitor of monoamine oxidase type B with potential for use in Parkinson's diseaseZreika M; Fozard J R; Dudley M W; Bey P; McDonald I A; Palfreyman M GJournal of neural transmission. Parkinson's disease and dementia section (1989), 1 (4), 243-54 ISSN:0936-3076.MDL 72,974, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine, was designed to be a selective inhibitor of monoamine oxidase type B (MAO-B). In vitro, the compound inhibits rat brain mitochondrial MAO in a concentration and time-dependent fashion and shows marked selectivity for the B form (IC50 = 680 and 3.6 nM for MAO-A and MAO-B, respectively). After oral administration to rats, the compound shows preferential inhibition of brain MAO-B with ED50 values of 8 and 0.18 mg/kg p.o. for the A and B forms, respectively. Selectivity is retained on repeat dosing. MDL 72,974 did not significantly potentiate the cardiovascular effects of intraduodenually-administered tyramine in anaesthetized rats and had only minor indirect sympathomimatic effects in the pithed rat. At MAO-B selective doses the neurotoxic effect of MPTP in mice was blocked.215Milczek, E. M.; Bonivento, D.; Binda, C.; Mattevi, A.; McDonald, I. A.; Edmondson, D. E. Structural and mechanistic studies of mofegiline inhibition of recombinant human monoamine oxidase B. J. Med. Chem. 2008, 51, 8019– 8026, DOI: 10.1021/jm8011867[ACS Full Text], [CAS], Google Scholar215https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVKlu73L&md5=a2155647066b1e971ecc2b7680ee7beeStructural and Mechanistic Studies of Mofegiline Inhibition of Recombinant Human Monoamine Oxidase BMilczek, Erika M.; Bonivento, Daniele; Binda, Claudia; Mattevi, Andrea; McDonald, Ian A.; Edmondson, Dale E.Journal of Medicinal Chemistry (2008), 51 (24), 8019-8026CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Mechanistic and structural studies have been carried out to investigate the mol. basis for the irreversible inhibition of human MAO-B by mofegiline. Competitive inhibition with substrate shows an apparent Ki of 28 nM. Irreversible inhibition of MAO-B occurs with a 1:1 M stoichiometry with no observable catalytic turnover. The absorption spectral properties of mofegiline inhibited MAO-B show features (λmax ≃ 450 nm) unlike those of traditional flavin N(5) or C(4a) adducts. Visible and near-UV CD spectra of the mofegiline-MAO-B adduct shows a neg. peak at 340 nm with an intensity similar to that of N(5) flavocyanine adducts. The x-ray crystal structure of the mofegiline-MAO-B adduct shows a covalent bond between the flavin cofactor N(5) with the distal allylamine carbon atom as well as the absence of the fluorine atom. A mechanism to explain these structural and spectral data is proposed.216Palfreyman, M. G.; McDonald, I. A.; Bey, P.; Danzin, C.; Zreika, M.; Cremer, G. Haloallylamine inhibitors of MAO and SSAO and their therapeutic potential. J. Neural. Transm. Suppl. 1994, 41, 407– 414, DOI: 10.1007/978-3-7091-9324-2_54[Crossref], [PubMed], [CAS], Google Scholar216https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXlsFalsbY%253D&md5=e3b1e61467c4cca735cb5f8d9e29e2c7Haloallylamine inhibitors of MAO and SSAO and their therapeutic potentialPalfreyman, M. G.; McDonald, I. A.; Bey, P.; Danzin, C.; Zreika, M.; Cremer, G.Journal of Neural Transmission, Supplement (1994), 41 (), 407-14CODEN: JNTSD4; ISSN:0303-6995.Based on mechanistic understandings, mol. modeling and extensive quant. structure-activity relationships, appropriately substituted haloallylamine derivs. were designed as potential mechanism-based inhibitors of MAO and/or semicarbazide-sensitive amine oxidase (SSAO). Potent inhibition of MAO-B and SSAO occurred with fluoroallylamines whereas chloroallylamines, such as MDL 72274A ((E)-2-phenyl-3-chloroallylamine hydrochloride), were selective and potent inhibitors of SSAO. MDL 72974A (E)-2-(4-fluorophenethyl)-3-fluoroallylamine hydrochloride is a potent (IC50 = 10-9M) inhibitor of both MAO-B and SSAO, with 190-fold lower affinity for MAO-A. In clin. studies, oral doses as low as 100 μg produced substantial inhibition of platelet MAO-B. Essentially complete inhibition occurred at 1 mg with the effect lasting 6-10 days. One or 4 mg MDL 72974A given daily for 28 days to 40 Parkinson's patients treated with L-dopa produced statistically significant redns. in the Unified Parkinson's Disease Rating Scale. MAO-B inhibitors, such as MDL 72974A and L-deprenyl, offer the potential of being neuroprotective in Parkinson's Disease and other neurogenerative disorders. Concomitant inhibition of SSAO may provide addnl., but as yet unproven, advantages over pure inhibitors of MAO-B.217Foot, J. S.; Deodhar, M.; Turner, C. I.; Yin, P.; van Dam, E. M.; Silva, D. G.; Olivieri, A.; Holt, A.; McDonald, I. A. The discovery and development of selective 3-fluoro-4-aryloxyallylamine inhibitors of the amine oxidase activity of semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1). Bioorg. Med. Chem. Lett. 2012, 22, 3935– 3940, DOI: 10.1016/j.bmcl.2012.04.111[Crossref], [PubMed], [CAS], Google Scholar217https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XntVyrtLg%253D&md5=cb02b80379ec2a2a6323b158f2297944The discovery and development of selective 3-fluoro-4-aryloxyallylamine inhibitors of the amine oxidase activity of semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1)Foot, Jonathan S.; Deodhar, Mandar; Turner, Craig I.; Yin, Ping; van Dam, Ellen M.; Silva, Diego G.; Olivieri, Aldo; Holt, Andrew; McDonald, Ian A.Bioorganic & Medicinal Chemistry Letters (2012), 22 (12), 3935-3940CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)A new class of 3-fluoroallyl amine-based SSAO/VAP-1 inhibitors is reported. These compds. have excellent selectivity over diamine oxidase, MAO-A and MAO-B. Synthesis and SAR studies leading to compd. 28 (PXS-4159A, I) are reported. The pharmacokinetic profile of 28 in the rat, together with activity in a murine model of lung inflammation are also disclosed.218Salmi, M.; Jalkanen, S. Cell-surface enzymes in control of leukocyte trafficking. Nat. Rev. Immunol. 2005, 5, 760– 771, DOI: 10.1038/nri1705[Crossref], [PubMed], [CAS], Google Scholar218https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtVGrurvF&md5=ca02d208fc611365ba8f7d7dcb3739c0Cell-surface enzymes in control of leukocyte traffickingSalmi, Marko; Jalkanen, SirpaNature Reviews Immunology (2005), 5 (10), 760-771CODEN: NRIABX; ISSN:1474-1733. (Nature Publishing Group)A review. Leukocyte trafficking between the blood and the tissues is pivotal for normal immune responses. Cell-adhesion mols. (such as selectins and leukocyte integrins) and chemoattractants (such as chemokines) have well-established roles in supporting leukocyte exit from the blood. Emerging data now show that, for both leukocytes and endothelial cells, enzymic reactions that are catalyzed by cell-surface-expressed enzymes with catalytic domains outside the plasma membrane (known as ectoenzymes) also make crucial contributions to this process. Ectoenzymes can function phys. as adhesion receptors and can regulate the recruitment of cells through their catalytic activities. Here, the authors provide new insights into how ectoenzymes - including nucleotidases, cyclases, ADP-ribosyltransferases, peptidases, proteases and oxidases - guide leukocyte traffic.219O’Rourke, A. M.; Wang, E. Y.; Miller, A.; Podar, E. M.; Scheyhing, K.; Huang, L.; Kessler, C.; Gao, H.; Ton-Nu, H. T.; Macdonald, M. T.; Jones, D. S.; Linnik, M. D. Anti-inflammatory effects of LJP 1586 [Z-3-fluoro-2-(4-methoxybenzyl)allylamine hydrochloride], an amine-based inhibitor of semicarbazide-sensitive amine oxidase activity. J. Pharmacol. Exp. Ther. 2008, 324, 867– 875, DOI: 10.1124/jpet.107.131672[Crossref], [PubMed], [CAS], Google Scholar219https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsFShurY%253D&md5=488fa2c75c5488de6cb038c1a253c72eAnti-inflammatory effects of LJP 1586 [Z-3-fluoro-2-(4-methoxybenzyl)allylamine hydrochloride], an amine-based inhibitor of semicarbazide-sensitive amine oxidase activityO'Rourke, Anne M.; Wang, Eric Y.; Miller, Andrew; Podar, Erika M.; Scheyhing, Kelly; Huang, Li; Kessler, Christina; Gao, Hongfeng; Ton-Nu, Huong-Thu; MacDonald, Mary T.; Jones, David S.; Linnik, Matthew D.Journal of Pharmacology and Experimental Therapeutics (2008), 324 (2), 867-875CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)Semicarbazide-sensitive amine oxidase (SSAO, amine oxidase, copper-contg. 3, and vascular adhesion protein-1) is a copper-contg. enzyme that catalyzes the oxidative deamination of primary amines to an aldehyde, ammonia, and hydrogen peroxide. SSAO is also involved in leukocyte migration to sites of inflammation, and the enzymic activity of SSAO is essential to this role. Thus, inhibition of SSAO enzyme activity represents a target for the development of small mol. anti-inflammatory compds. Here, we have characterized the novel SSAO inhibitor, Z-3-fluoro-2-(4-methoxybenzyl)allylamine hydrochloride (LJP 1586), and assessed its anti-inflammatory activity. LJP 1586 is a potent inhibitor of rodent and human SSAO activity, with IC50 values between 4 and 43 nM. The selectivity of LJP 1586 was confirmed with a broad panel of receptors and enzymes that included the monoamine oxidases A and B. Oral administration of LJP 1586 resulted in complete inhibition of rat lung SSAO, with an ED50 between 0.1 and 1 mg/kg, and a pharmacodynamic half-life of greater than 24 h. In a mouse model of inflammatory leukocyte trafficking oral dosing with LJP 1586 resulted in significant dose-dependent inhibition of neutrophil accumulation, with an effect comparable to that of anti-leukocyte function-assocd. antigen-1 antibody. In a rat model of LPS-induced lung inflammation, administration of 10 mg/kg LJP 1586 resulted in a 55% significant redn. in transmigrated cells recovered by bronchoalveolar lavage. The results demonstrate that a selective, orally active small mol. inhibitor of SSAO is an effective anti-inflammatory compd. in vivo and provide further support for SSAO as a therapeutic anti-inflammatory target.220Dow, J.; Piriou, F.; Wolf, E.; Dulery, B. D.; Haegele, K. D. Novel carbamate metabolites of mofegiline, a primary amine monoamine oxidase B inhibitor, in dogs and humans. Drug Metab. Dispos. 1994, 22, 738– 749[PubMed], [CAS], Google Scholar220https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXmslWhs78%253D&md5=b437862f1906cba7f30bbc0a633c3ff6Novel carbamate metabolites of mofegiline, a primary amine monoamine oxidase B inhibitor, in dogs and humansDow, James; Piriou, Francois; Wolf, Evelyne; Dulery, Bertrand D.; Haegele, Klaus D.Drug Metabolism and Disposition (1994), 22 (5), 738-49CODEN: DMDSAI; ISSN:0090-9556.Mofegiline or MDL 72,974A ((E)-4-fluoro-β-fluoromethylene benzene butanamine hydrochloride) is a selective enzyme-activated irreversible inhibitor of monoamine oxidase B, which is under development for use in the treatment of Parkinson's disease. Male beagle dogs were given single oral (20 mg/kg) and i.v. (5 mg/kg) doses of [14C]-Mofegiline. Total radioactivity excreted in urine and feces over 96 h was, resp., 75.5 and 6.3% of the dose after oral and 67.9 and 3.9% after i.v. administration. Unchanged drug in urine represented 3% of the dose after oral and less than 1% after i.v. administration. Mofegiline was thus extensively metabolized in dogs, and urinary excretion was the major route of elimination of metabolites. HPLC, with online radioactivity detection, showed the presence of four major peaks (M1, M2, M3, and M4), representing resp. 50, 9, 5, and 0.5% of the administered dose excreted in 0.24 h urine. TSP-LC-MS, Fab-MS, and NMR spectra of the purified metabolites were obtained. M1, the major metabolite in dogs, was shown to have undergone defluorination of the β-fluoromethylene moiety, and one carbon addn. Its structure was confirmed to be a cyclic carbamate. M2 was a N-carbamoyl-O-β-D-glucuronide conjugate of parent drug. The formation of M1 and M2 is likely to involve initial reversible addn. of CO2 to the primary amine function. M3 was a N-succinyl conjugate of the parent drug. M4 had also undergone defluorination to yield a urea adduct of an unsatd. α,β aldehyde. Structures of M1 and M3 were further confirmed by comparing their MS and NMR spectra with those of authentic ref. compds. TSP-LC-MS ion chromatographs of human urine, obtained from two male volunteers after oral administration of 24 mg of drug, showed selected mol. ion peaks with the same retention time as the metabolites identified in dogs. In humans, these common metabolites represented a similar percentage of the administered dose to that in dogs. The present study demonstrates that NMR, TSP-LC-MS, and FAB-MS are complementary anal. techniques, which allow structural identification of unhydrolyzed drug conjugates. The formation of carbamates of amine-contg. drugs may be more common than previously reported.221Pan, Y.; Gerasimov, M. R.; Kvist, T.; Wellendorph, P.; Madsen, K. K.; Pera, E.; Lee, H.; Schousboe, A.; Chebib, M.; Brauner-Osborne, H.; Craft, C. M.; Brodie, J. D.; Schiffer, W. K.; Dewey, S. L.; Miller, S. R.; Silverman, R. B. (1S, 3S)-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (CPP-115), a potent γ-aminobutyric acid aminotransferase inactivator for the treatment of cocaine addiction. J. Med. Chem. 2012, 55, 357– 366, DOI: 10.1021/jm201231w[ACS Full Text], [CAS], Google Scholar221https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFGmt7jN&md5=70944b375478c340c8d0c999317b4293(1S, 3S)-3-Amino-4-difluoromethylenyl-1-cyclopentanoic Acid (CPP-115), a Potent γ-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Cocaine AddictionPan, Yue; Gerasimov, Madina R.; Kvist, Trine; Wellendorph, Petrine; Madsen, Karsten K.; Pera, Elena; Lee, Hyunbeom; Schousboe, Arne; Chebib, Mary; Brauner-Osborne, Hans; Craft, Cheryl M.; Brodie, Jonathan D.; Schiffer, Wynne K.; Dewey, Stephen L.; Miller, Steven R.; Silverman, Richard B.Journal of Medicinal Chemistry (2012), 55 (1), 357-366CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Vigabatrin, a GABA aminotransferase (GABA-AT) inactivator, is used to treat infantile spasms and refractory complex partial seizures and is in clin. trials to treat addiction. We evaluated a novel GABA-AT inactivator (1S, 3S)-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (CPP-115, compd. 1) and obsd. that it does not exhibit other GABAergic or off-target activities and is rapidly and completely orally absorbed and eliminated. By use of in vivo microdialysis techniques in freely moving rats and microPET imaging techniques, 1 produced similar inhibition of cocaine-induced increases in extracellular dopamine and in synaptic dopamine in the nucleus accumbens at 1/300 to 1/600 the dose of vigabatrin. It also blocks expression of cocaine-induced conditioned place preference at a dose 1/300 that of vigabatrin. Electroretinog. (ERG) responses in rats treated with 1, at doses 20-40 times higher than those needed to treat addiction in rats, exhibited redns. in ERG responses, which were less than the redns. obsd. in rats treated with vigabatrin at the same dose needed to treat addiction in rats. In conclusion, 1 can be administered at significantly lower doses than vigabatrin, which suggests a potential new treatment for addiction with a significantly reduced risk of visual field defects.222Silverman, R. B. Design and mechanism of GABA aminotransferase inactivators. Treatments for epilepsies and addictions. Chem. Rev. 2018, 118, 4037– 4070, DOI: 10.1021/acs.chemrev.8b00009[ACS Full Text], [CAS], Google Scholar222https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXlt1yhu7k%253D&md5=0071d0e7668f05d37db995bb10430c16Design and Mechanism of GABA Aminotransferase Inactivators. Treatments for Epilepsies and AddictionsSilverman, Richard B.Chemical Reviews (Washington, DC, United States) (2018), 118 (7), 4037-4070CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. When the brain concn. of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) diminishes below a threshold level, the excess neuronal excitation can lead to convulsions. This imbalance in neurotransmission can be cor. by inhibition of the enzyme γ-aminobutyric acid aminotransferase (GABA-AT), which catalyzes the conversion of GABA to the excitatory neurotransmitter L-glutamic acid. It also has been found that raising GABA levels can antagonize the rapid elevation and release of dopamine in the nucleus accumbens, which is responsible for the reward response in addiction. Therefore, the design of new inhibitors of GABA-AT, which increases brain GABA levels, is an important approach to new treatments for epilepsy and addiction. This review summarizes findings over the last 40 or so years of mechanism-based inactivators (unreactive compds. that require the target enzyme to catalyze their conversion to the inactivating species, which inactivate the enzyme prior to their release) of GABA-AT with emphasis on their catalytic mechanisms of inactivation, presented according to org. chem. mechanism, with minimal pharmacol., except where important for activity in epilepsy and addiction. Patents, abstrs., and conference proceedings are not covered in this review. The inactivation mechanisms described here can be applied to the inactivations of a wide variety of unrelated enzymes.223Lippert, B.; Metcalf, B. W.; Jung, M. J.; Casara, P. 4-Amino-hex-5-enoic acid, a selective catalytic inhibitor of 4-aminobutyric-acid aminotransferase in mammalian brain. Eur. J. Biochem. 1977, 74, 441– 445, DOI: 10.1111/j.1432-1033.1977.tb11410.x[Crossref], [PubMed], [CAS], Google Scholar223https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2sXktVWlu7o%253D&md5=849abfb7d249e7e40457c38e3ddfb0ff4-Amino-hex-5-enoic acid, a selective catalytic inhibitor of 4-aminobutyric-acid aminotransferase in mammalian brainLippert, Bruce; Metcalf, Brian W.; Jung, Michel J.; Casara, PatrickEuropean Journal of Biochemistry (1977), 74 (3), 441-5CODEN: EJBCAI; ISSN:0014-2956.Incubation of rat brain 4-aminobutyrate (I) aminotransferase (II) with 4-amino-hex-5-enoic acid (III), an analog of I, results in a time-dependent irreversible loss of activity. In the presence of 0.1 mM III, the half-life of the inactivation process is approx. 6 min. Low concns. of L-glutamic acid or I protect against this inactivation, while 2-oxoglutarate prevents this protection, suggesting that only the pyridoxal form of II is susceptible to inhibition by III. The irreversible inhibition of mammalian II by III is selective. There is no inhibition of II from Pseudomonas fluorescens with III at mM concns. Even at 10 mM, there is no irreversible inhibition of mammalian glutamate decarboxylase or of aspartate aminotransferase, while alanine aminotransferase is inhibited >500-fold more slowly than II.224Storici, P.; De Biase, D.; Bossa, F.; Bruno, S.; Mozzarelli, A.; Peneff, C.; Silverman, R. B.; Schirmer, T. Structures of γ-aminobutyric acid (GABA) aminotransferase, a pyridoxal 5′-phosphate, and [2Fe-2S] cluster-containing enzyme, complexed with γ-ethynyl-GABA and with the antiepilepsy drug vigabatrin. J. Biol. Chem. 2004, 279, 363– 373, DOI: 10.1074/jbc.M305884200[Crossref], [PubMed], [CAS], Google Scholar224https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhtVSqtrfK&md5=5c0b22682c6419f86551270df45cb6d7Structures of γ-aminobutyric acid (GABA) aminotransferase, a pyridoxal 5'-phosphate, and [2Fe-2S] cluster-containing enzyme, complexed with γ-ethynyl-GABA and with the antiepilepsy drug vigabatrinStorici, Paola; De Biase, Daniela; Bossa, Francesco; Bruno, Stefano; Mozzarelli, Andrea; Peneff, Caroline; Silverman, Richard B.; Schirmer, TilmanJournal of Biological Chemistry (2004), 279 (1), 363-373CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)γ-Aminobutyrate (GABA) aminotransferase (I) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme responsible for the degrdn. of the inhibitory neurotransmitter, GABA. I is a validated target for antiepilepsy drugs because its selective inhibition raises GABA concns. in brain. The antiepilepsy drug, vigabatrin (γ-vinyl-GABA), was investigated in the past by various biochem. methods and these resulted in several proposals for its mechanisms of inactivation. Here, the authors solved and compared the crystal structures of pig liver I in its native form (to 2.3-Å resoln.) and its complex with vigabatrin as well as with its close analog, γ-ethynyl-GABA (to 2.3 and 2.8 Å, resp.). Both inactivators formed a covalent ternary adduct with the active site Lys-329 residue and the PLP cofactor. The crystal structures provided direct support for specific inactivation mechanisms proposed earlier on the basis of radiolabeling expts. The reactivity of I crystals with the 2 GABA analogs was also investigated by polarized absorption microspectrophotometry. The spectral data were discussed in relation to the proposed mechanism. Intriguingly, all 3 structures revealed a [2Fe-2S] cluster of yet unknown function at the center of the dimeric mol. in the vicinity of the PLP cofactor.225Pan, Y.; Qiu, J.; Silverman, R. B. Design, synthesis, and biological activity of a difluoro-substituted, conformationally rigid vigabatrin analogue as a potent γ-aminobutyric acid aminotransferase inhibitor. J. Med. Chem. 2003, 46, 5292– 5293, DOI: 10.1021/jm034162s[ACS Full Text], [CAS], Google Scholar225https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXovVClt7k%253D&md5=30a2fb61e2beb595154f7a9519188d63Design, Synthesis, and Biological Activity of a Difluoro-Substituted, Conformationally Rigid Vigabatrin Analogue as a Potent γ-Aminobutyric Acid Aminotransferase InhibitorPan, Yue; Qiu, Jian; Silverman, Richard B.Journal of Medicinal Chemistry (2003), 46 (25), 5292-5293CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Previously it was found that a conformationally rigid analog [(1R,4S)-4-amino-2-cyclopentene-1-carboxylic acid] of the epilepsy drug (S)-vigabatrin did not inactivate γ-aminobutyric acid aminotransferase (GABA-AT). Ring opening of (1S,4S)-6-methylene-2-(phenylmethyl)-2-azabicyclo[2.2.1]heptan-3-one gave (1S,4S)-4-amino-3-methylene-1-cyclopentanecarboxylic acid (I). I, having an exocyclic double bond, was found to inactivate GABA-AT, but only in the absence of 2-mercaptoethanol. The corresponding difluoro-substituted analog [(+)-(1S,4S)-4-amino-3-(difluoromethylene)-1-cyclopentanecarboxylic acid] was synthesized and was shown to be a very potent time-dependent inhibitor, even in the presence of 2-mercaptoethanol.226Lee, H.; Doud, E. H.; Wu, R.; Sanishvili, R.; Juncosa, J. I.; Liu, D.; Kelleher, N. L.; Silverman, R. B. Mechanism of inactivation of γ-aminobutyric acid aminotransferase by (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115). J. Am. Chem. Soc. 2015, 137, 2628– 2640, DOI: 10.1021/ja512299n[ACS Full Text], [CAS], Google Scholar226https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVWgt7k%253D&md5=518c957dfdcd01be2a8f3b2e3ab2f14aMechanism of inactivation of γ-aminobutyric acid aminotransferase by (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115)Lee, Hyunbeom; Doud, Emma H.; Wu, Rui; Sanishvili, Ruslan; Juncosa, Jose I.; Liu, Dali; Kelleher, Neil L.; Silverman, Richard B.Journal of the American Chemical Society (2015), 137 (7), 2628-2640CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)γ-Aminobutyrate (GABA) aminotransferase (I) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concn. of GABA falls below a threshold level, convulsions can occur. Inhibition of I raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurol. disorders, including drug addiction. Among the analogs that the authors previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115), showed 187-fold greater potency than that of vigabatrin, a known inactivator of I and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently, CPP-115 was shown to have no adverse effects in a phase I clin. trial. Here, the authors report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes I-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of 2 F- ions and coenzyme conversion to pyridoxamine 5'-phosphate (PMP). The partition ratio for CPP-115 with pig brain I was ∼2000, releasing cyclopentanone-2,4-dicarboxylateand 2 other precursors of this compd. Time-dependent inactivation occurred by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP, which disrupted an electrostatic interaction between Glu-270 and Arg-445 to form an electrostatic interaction between Arg-445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. This represents a novel mechanism for inactivation of I and a new approach for the design of mechanism-based inactivators in general.227Jeschke, P.; Baston, E.; Leroux, F. R. α-Fluorinated ethers as “exotic” entity in medicinal chemistry. Mini-Rev. Med. Chem. 2007, 7, 1027– 1034, DOI: 10.2174/138955707782110150[Crossref], [PubMed], [CAS], Google Scholar227https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1Sjs7fN&md5=ad28d99858f35257c9bec151e6a4db9aα-Fluorinated ethers as "exotic" entity in medicinal chemistryJeschke, Peter; Baston, Eckhard; Leroux, Frederic R.Mini-Reviews in Medicinal Chemistry (2007), 7 (10), 1027-1034CODEN: MMCIAE; ISSN:1389-5575. (Bentham Science Publishers Ltd.)A review. After nitrogen, fluorine occupies the position of second favorite heteroelement in life science-oriented research. In contrast, the trifluoromethoxy group is still perhaps the least well understood fluorine substituent, although its occurrence has significantly increased in the recent years. Today, significant application areas for trifluoromethoxy substituted pharmaceuticals are in the field of analgesics, anesthetics, cardiovascular drugs, respiratory drugs, psychopharmacol. drugs, neurol. drugs, gastrointestinal drugs and anti-infective therapeutics. The present review will give an overlook of its use in medicinal chem.228Logvinenko, I. G.; Markushyna, Y.; Kondratov, I. S.; Vashchenko, B. V.; Kliachyna, M.; Tokaryeva, Y.; Pivnytska, V.; Grygorenko, O. O.; Haufe, G. Synthesis, physico-chemical properties and microsomal stability of compounds bearing aliphatic trifluoromethoxy group. J. Fluorine Chem. 2020, 231, 109461, DOI: 10.1016/j.jfluchem.2020.109461[Crossref], [CAS], Google Scholar228https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXivVens7c%253D&md5=283bf1da7c5138c4a6d4b7df6eb8bdb6Synthesis, physico-chemical properties and microsomal stability of compounds bearing aliphatic trifluoromethoxy groupLogvinenko, Ivan G.; Markushyna, Yevheniia; Kondratov, Ivan S.; Vashchenko, Bohdan V.; Kliachyna, Maria; Tokaryeva, Yuliya; Pivnytska, Valentyna; Grygorenko, Oleksandr O.; Haufe, GunterJournal of Fluorine Chemistry (2020), 231 (), 109461CODEN: JFLCAR; ISSN:0022-1139. (Elsevier B.V.)Effects of the trifluoromethoxy substituent on physico-chem. properties of compds., such as kinetic soly., lipophilicity and microsomal clearance, was studied in a series of aliph. derivs. It was found that kinetic soly. of the CF3O-contg. compds. was comparable to that of analogs, i.e. compds. bearing CH3O and CF3 moieties. The CF3O-substituted compds. had higher lipophilicity as compared to methoxy analogs, and nearly the same like CF3-bearing compds. Microsomal stability studies indicated that the trifluoromethoxy group typically decreased metabolic stability of the corresponding derivs. as compared to either CH3O- or CF3-substituted counterparts, except for N-alkoxy(sulfon)amide series.229Trachsel, D. Fluorine in psychedelic phenethylamines. Drug Test. Anal. 2012, 4, 577– 590, DOI: 10.1002/dta.413[Crossref], [PubMed], [CAS], Google Scholar229https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XivVGhu7s%253D&md5=a9609e3ef07f65c2bb5778d04352bf6cFluorine in psychedelic phenethylaminesTrachsel, DanielDrug Testing and Analysis (2012), 4 (7-8), 577-590CODEN: DTARBG; ISSN:1942-7603. (John Wiley & Sons Ltd.)A review. The so-called psychedelic phenethylamines represent a class of drugs with a large range of psychoactive properties in humans, ranging from naturally occurring mescaline to amphetamine analogs and homologues. The interest in many of these compds., occasionally referred to as designer-drugs, is widely dispersed across popular culture and political and scientific communities. In recent decades, fluorine has become a powerful and important tool in medicinal chem. In addn., fluorine-contg. compds. and medicines can be found in numerous com. successful pharmaceuticals that have gained a market share of some 5-15%. One might anticipate this trend to increase in the future. As far as fluorinated phenethylamines are concerned, much less is known about their chem. and pharmacol. This paper provides an overview regarding the biol. properties of over 60 fluorinated phenethylamines and discusses both historical and recent chem.-related developments. It was shown that the introduction of fluorine into the phenethylamine nucleus can impact greatly on psychoactivity of these compds., ranging from marked loss to enhancement and prolongation of effects. For example, in contrast to the psychoactive escaline (70), it was obsd. that its fluoroescaline (76) counterpart was almost devoid of psychoactive effects. Difluoroescaline (77), on the other hand, retained, and trifluoroescaline (78) showed increased human potency of escaline (70). Difluoromescaline (72) and trifluoromescaline (73) increasingly surpassed human potency and duration of mescaline (22) effects. Copyright © 2012 John Wiley & Sons, Ltd.230Laurence, C.; Brameld, K. A.; Graton, J.; Le Questel, J. Y.; Renault, E. The pK(BHX) database: toward a better understanding of hydrogen-bond basicity for medicinal chemists. J. Med. 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C.; Narayanan, A.; Unwalla, R.; Lovering, F.; Denny, R. A.; Zhou, H.; Bunnage, M. E. Fluorine in drug design: a case study with fluoroanisoles. ChemMedChem 2015, 10, 715– 726, DOI: 10.1002/cmdc.201402555[Crossref], [PubMed], [CAS], Google Scholar231https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkvVWku7Y%253D&md5=45dbead703594c31581cc6c7e62d1b81Fluorine in Drug Design: A Case Study with FluoroanisolesXing, Li; Blakemore, David C.; Narayanan, Arjun; Unwalla, Ray; Lovering, Frank; Denny, R. Aldrin; Zhou, Huanyu; Bunnage, Mark E.ChemMedChem (2015), 10 (4), 715-726CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)Anisole and fluoroanisoles display distinct conformational preferences, as evident from a survey of their crystal structures. In addn. to altering the free ligand conformation, various degrees of fluorination have a strong impact on physicochem. and pharmacokinetic properties. Anal. of anisole and fluoroanisole matched mol. pairs in the Pfizer corporate database reveals interesting trends: (1) PhOCF3 increases log D by ∼1 log unit over PhOCH3 compds.; (2) PhOCF3 shows lower passive permeability despite its higher lipophilicity; and (3) PhOCF3 does not appreciably improve metabolic stability over PhOCH3. Emerging from the investigation, difluoroanisole (PhOCF2H) strikes a better balance of properties with noticeable advantages of log D and transcellular permeability over PhOCF3. Synthetic assessment illustrates that the routes to access difluoroanisoles are often more straightforward than those for trifluoroanisoles. Whereas replacing PhOCH3 with PhOCF3 is a common tactic to optimize ADME properties, the anal. suggests PhOCF2H may be a more attractive alternative, and greater exploitation of this motif is recommended.232Federsel, D.; Herrmann, A.; Christen, D.; Sander, S.; Willner, H.; Oberhammer, H. Structure and conformation of α,α,α-trifluoroanisol, C6H5OCF3. J. Mol. Struct. 2001, 567–568, 127– 136, DOI: 10.1016/S0022-2860(01)00541-5[Crossref], [CAS], Google Scholar232https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXlvF2isb0%253D&md5=4b1903e5580b8440c797d4e9031d5da4Structure and conformation of α,α,α-trifluoroanisole, C6H5OCF3Federsel, Daniela; Herrmann, Angelika; Christen, Dines; Sander, Stefan; Willner, Helge; Oberhammer, HeinzJournal of Molecular Structure (2001), 567-568 (), 127-136CODEN: JMOSB4; ISSN:0022-2860. (Elsevier Science B.V.)The geometric structure of α,α,α-trifluoroanisole, C6H5OCF3, was studied by gas electron diffraction (GED), microwave spectroscopy (MW), matrix IR spectroscopy and quantum chem. methods. From the three exptl. techniques, we conclude that only the perpendicular conformer (O-CF3 bond perpendicular to the benzene plane) is present in the gas-phase, although a slightly better fit of the GED intensities is obtained for a small contribution (12(8)%) of the planar form. Thus, the orientation of the O-CH3 bond in anisole changes from planar to perpendicular upon fluorination of the Me group. The predictions of quantum chem. calcns. for the conformational properties depend on the computational method and on the size of basis sets. HF/6-31G* and MP2 (6-31G* or 6-311(2d) basis sets) calcns. predict the existence of a single conformer with perpendicular orientation, in agreement with our exptl. data. The B3LYP method, however, results in a mixt. of both conformers and HF/3-21G* calcns. predict a single conformer with intermediate orientation.233Klocker, J.; Karpfen, A.; Wolschann, P. On the structure and torsional potential of trifluoromethoxybenzene: an ab initio and density functional study. Chem. Phys. Lett. 2003, 367, 566– 575, DOI: 10.1016/S0009-2614(02)01786-4[Crossref], [CAS], Google Scholar233https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XptlWgt70%253D&md5=bb476890fdb2a1e48ffdbe9966d8515aOn the structure and torsional potential of trifluoromethoxybenzene: an ab initio and density functional studyKlocker, Johanna; Karpfen, Alfred; Wolschann, PeterChemical Physics Letters (2002), 367 (5,6), 566-575CODEN: CHPLBC; ISSN:0009-2614. (Elsevier Science B.V.)The torsional potential of trifluoromethoxybenzene around the aryl-O bond was investigated with the aid of large-scale ab initio calcns. performed at the Moller-Plesset second order (MP2) level, with several post-MP2 methods, and with a hybrid d. functional method (B3LYP). Contrary to several recent reports, we do not find substantial qual. differences between MP2 and B3LYP results, provided sufficiently large basis sets are used. The results are confronted with analogous MP2 and B3LYP data for methoxybenzene, for hypothetical anions as obtained by deprotonation at the para-position, and for ethylbenzene. The trends in the calcd. torsional potentials, barrier heights and energy differences between conformers are discussed and correlated with selected structural parameters.234Kuo, E. A.; Hambleton, P. T.; Kay, D. P.; Evans, P. L.; Matharu, S. S.; Little, E.; McDowall, N.; Jones, C. B.; Hedgecock, C. J.; Yea, C. M.; Chan, A. W.; Hairsine, P. W.; Ager, I. R.; Tully, W. R.; Williamson, R. A.; Westwood, R. Synthesis, structure-activity relationships, and pharmacokinetic properties of dihydroorotate dehydrogenase inhibitors: 2-cyano-3-cyclopropyl-3-hydroxy-N-[3′-methyl-4’-(trifluoromethyl)phenyl ] propenamide and related compounds. J. Med. Chem. 1996, 39, 4608– 4621, DOI: 10.1021/jm9604437[ACS Full Text], [CAS], Google Scholar234https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xmt1ylsLo%253D&md5=1bffdde8bb8429fd7d309f7bb26660bcSynthesis, Structure-Activity Relationships, and Pharmacokinetic Properties of Dihydroorotate Dehydrogenase Inhibitors: 2-Cyano-3-cyclopropyl-3-hydroxy- N-[3'-methyl-4'-(trifluoromethyl)phenyl]propenamide and Related CompoundsKuo, Elizabeth A.; Hambleton, Philip T.; Kay, David P.; Evans, Phillip L.; Matharu, Saroop S.; Little, Edward; McDowall, Neil; Jones, C. Beth; Hedgecock, Charles J. R.; et al.Journal of Medicinal Chemistry (1996), 39 (23), 4608-4621CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The active, ring-opened metabolite of the novel immunosuppressive agent leflunomide has been shown to inhibit the enzyme dihydroorotate dehydrogenase (DHODH). This enzyme catalyzes the fourth step in de novo pyrimidine biosynthesis. A series of analogs of the leflunomide metabolite have been synthesized. Their in vivo biol. activity detd. in rat and mouse delayed type hypersensitivity has been found to correlate well with their in vitro DHODH potency. The most promising compd. has shown activity in rat and mouse collagen (II)-induced arthritis models (ED50 = 2 and 31 mg/kg, resp.) and has shown a shorter half-life in man when compared with leflunomide. Clin. studies in rheumatoid arthritis are in progress.235Dihel, L.; Kittleson, C.; Mulvihill, K.; Johnson, W. W. Oxidative metabolism of the trifluoromethoxy moiety of OSI-930. Drug Metab. Drug Interact. 2009, 24, 95– 121, DOI: 10.1515/DMDI.2009.24.2-4.95[Crossref], [PubMed], [CAS], Google Scholar235https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXktlOqu7o%253D&md5=8896ae50e1ffda023f083637e4bf859dOxidative metabolism of the trifluoromethoxy moiety of OSI-930Dihel, Larry; Kittleson, Christine; Mulvihill, Kristen; Johnson, William W.Drug Metabolism and Drug Interactions (2009), 24 (2-4), 95-121CODEN: DMDIEQ; ISSN:0792-5077. (Freund Publishing House Ltd.)Cytochrome P 450 can catalyze a wide array of remarkable oxidns., including O-dealkylations, which are performed via oxidn. of the α-carbon of the ether. When C-H bonds are replaced with C-F bonds, however, the bond strength is much greater, and it significantly deters oxidn. at the carbon. Another recently elucidated reaction catalyzed by P 450, ipso substitution, results in displacement of arom. ring substituents such as an alkoxy group via hydroxyl substitution. Through LC/MS/MS, we show the CYP-mediated oxidative displacement of the trifluoromethoxy group from the Ph constituent in OSI-930, a novel small mol. c-Kit/VEGF-r inhibitor in clin. studies to treat cancer. Based on C-F bond strength, reported phenacetin studies, and α-quaternary alkylphenol studies, we propose an ipso-substitution mechanism for this oxidative biotransformation. In vivo, this hydroxylated metabolite goes on to form the ether conjugate with glucuronide.236Hinson, J. A.; Nelson, S. D.; Gillette, J. R. Metabolism of [p-18O]-phenacetin: the mechanism of activation of phenacetin to reactive metabolites in hamsters. Mol. Pharmacol. 1979, 15, 419– 427[PubMed], [CAS], Google Scholar236https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1MXktlamur0%253D&md5=282d8fcd9d48a3d65597a407f3baafb7Metabolism of [p-18O]-phenacetin: the mechanism of activation of phenacetin to reactive metabolites in hamstersHinson, Jack A.; Nelson, Sidney D.; Gillette, James R.Molecular Pharmacology (1979), 15 (2), 419-27CODEN: MOPMA3; ISSN:0026-895X.There are 3 different pathways by which phenacetin (I) [62-44-2] can be converted by liver enzymes to electrophilic reactive metabolites: (1) I is first deethylated to acetaminophen [103-90-2] which is subsequently activated by cytochrome P-450 [9035-51-2], (2) I is activated via an intermediate, (3) I is first converted to N-hydroxyphenacetin [19315-64-1], then activated by sulfation or glucuronidation. These 3 pathways were distinguished by the disposition of 18O in the para position of the ring; (1) when acetaminophen-4-18O was activated in vitro and in vivo there was a negligible loss of 18O in the resp. acetaminophen-glutathione conjugate and the urinary mercapturic acid [616-91-1]; (2) when I-4-18O wa activated in vitro there was a 50% loss of 18O in the acetaminophen-glutathione conjugate; (3) when N-hydroxyphenacetin glucuronide [69783-19-3] was incubated with H218O there was a quant. incorporation of 18O into the acetaminophen-glutathione conjugate. When N-hydroxyacetaminophen [70110-93-9], the proposed intermediate in pathway 1, was incubated with glutathione and H218O there was no incorporation of 18O into the acetaminophen-glutathione conjugate. The relative in vivo importance of the 3 pathways was investigated by i.p. injection of 50 mg/kg of I-4-18O into hamsters. The urinary mercapturic acid showed ∼10% loss of 18O label, indicating that deethylation of I to acetaminophen followed by activation of acetaminophen. Thus pathway 1 appears to be the predominant pathway in vivo.237Rietjens, I. M.; den Besten, C.; Hanzlik, R. P.; van Bladeren, P. J. Cytochrome P450-catalyzed oxidation of halobenzene derivatives. Chem. Res. Toxicol. 1997, 10, 629– 635, DOI: 10.1021/tx9601061[ACS Full Text], [CAS], Google Scholar237https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXjs1Wgt74%253D&md5=54b2699f7df748a5a507007040bf94adCytochrome P450-Catalyzed Oxidation of Halobenzene DerivativesRietjens, Ivonne M. C. M.; den Besten, Cathaline; Hanzlik, Robert P.; van Bladeren, Peter J.Chemical Research in Toxicology (1997), 10 (6), 629-635CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)A review and discussion with refs. Multiple pathways in the generation of (proximate) toxic metabolites of a halobenzene, alkylating properties of epoxides and quinones, mechanism of benzoquinone formation, role of epoxides in the pathway to phenolic metabolites are discussed.238National Library of Medicine. https://pubchem.ncbi.nlm.nih.gov/compound/Carbonyl-fluoride (accessed 2020-03-14).239Deinum, J.; Mattsson, C.; Inghardt, T.; Elg, M. Biochemical and pharmacological effects of the direct thrombin inhibitor AR-H067637. Thromb. Haemostasis 2009, 101, 1051– 1059, DOI: 10.1160/TH08-09-0586[Crossref], [PubMed], [CAS], Google Scholar239https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXos1aktbo%253D&md5=01ec6f363cff05b7389618e4e406a8eaBiochemical and pharmacological effects of the direct thrombin inhibitor AR-H067637Deinum, Johanna; Mattsson, Christer; Inghardt, Tord; Elg, MargaretaThrombosis and Haemostasis (2009), 101 (6), 1051-1059CODEN: THHADQ; ISSN:0340-6245. (Schattauer GmbH)AZD0837 is in development as a new oral anticoagulant for use in thromboembolic disorders. In vivo, AZD0837 is converted to AR-H067637, a selective and reversible direct thrombin inhibitor. Established biochem. methods were used to assess and measure the biochem. and pharmacol. properties of AR-H067637. Both direct Biacore binding studies of AR-H067637 with immobilized α-thrombin and inhibition studies using pre-steady state kinetics with thrombin in the fluid phase confirmed that AR-H067637 is a rapid-binding, reversible and potent (inhibition const. Ki = 2-4 nM), competitive inhibitor of thrombin, as well as of thrombin bound to fibrin (clotbound thrombin) or to thrombomodulin. The total amt. of free thrombin generated in platelet-poor clotting plasma was inhibited concn.-dependently by AR-H067637, with a concn. giving half maximal inhibition (IC50) of 0.6 μM. Moreover, AR-H067637 is, with the exception of trypsin, a selective inhibitor for thrombin without inhibiting other serine proteases involved in haemostasis. Furthermore, no anticoagulant effect of the prodrug was found AR-H067637 prolonged the clotting time concn.-dependently in a range of plasma coagulation assays including activated partial thromboplastin time, prothrombin time, prothrombinase-induced clotting time, thrombin time and ecarin clotting time. The two latter assays were found to be most sensitive for assessing the anticoagulant effect of AR-H067637 (plasma IC50 93 and 220 nM, resp.). AR-H067637 also inhibited thrombin-induced platelet activation (by glycoprotein Ilb/Illa exposure, IC50 8.4 nM) and aggregation (IC50 0.9 nM). In conclusion, AR-H067637 is a selective, reversible, competitive inhibitor of α-thrombin, with a predictable anticoagulant effect demonstrated in plasma coagulation assays.240Malamas, M. S.; Robichaud, A.; Erdei, J.; Quagliato, D.; Solvibile, W.; Zhou, P.; Morris, K.; Turner, J.; Wagner, E.; Fan, K.; Olland, A.; Jacobsen, S.; Reinhart, P.; Riddell, D.; Pangalos, M. Design and synthesis of aminohydantoins as potent and selective human β-secretase (BACE1) inhibitors with enhanced brain permeability. Bioorg. Med. Chem. Lett. 2010, 20, 6597– 6605, DOI: 10.1016/j.bmcl.2010.09.029[Crossref], [PubMed], [CAS], Google Scholar240https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlSmsL7I&md5=848a1bfa79278b0c7a72749f9be45ad8Design and synthesis of aminohydantoins as potent and selective human β-secretase (BACE1) inhibitors with enhanced brain permeabilityMalamas, Michael S.; Robichaud, Albert; Erdei, Jim; Quagliato, Dominick; Solvibile, William; Zhou, Ping; Morris, Koi; Turner, Jim; Wagner, Erik; Fan, Kristi; Olland, Andrea; Jacobsen, Steve; Reinhart, Peter; Riddell, David; Pangalos, MenelasBioorganic & Medicinal Chemistry Letters (2010), 20 (22), 6597-6605CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)The identification of small mol. aminohydantoins as potent and selective human β-secretase inhibitors is reported. These analogs exhibit good brain permeability (40-70%), low nanomolar potency for BACE1, and demonstrate >100-fold selectivity for the structurally related aspartyl proteases cathepsin D, renin and pepsin. Alkyl and alkoxy groups at the meta-position of the P1 Ph, which extend toward the S3 region of the enzyme, have contributed to the ligand's reduced affinity for the efflux transporter protein P-gp, and decreased topol. polar surface area, thus resulting in enhanced brain permeability. A fluorine substitution at the para-position of the P1 Ph has contributed to 100-fold decrease of CYP3A4 inhibition and enhancement of compd. metabolic stability. The plasma and brain protein binding properties of these new analogs are affected by substitutions at the P1 Ph moiety. Higher compd. protein binding was obsd. in the brain than in the plasma. Two structurally diverse potent BACE1 inhibitors (84 and 89) reduced 30% plasma Aβ40 in the Tg2576 mice in vivo model at 30 mg/kg po.241Williams, S. J.; Zammit, S. C.; Cox, A. J.; Shackleford, D. M.; Morizzi, J.; Zhang, Y.; Powell, A. K.; Gilbert, R. E.; Krum, H.; Kelly, D. J. 3′,4’-Bis-difluoromethoxycinnamoylanthranilate (FT061): an orally-active antifibrotic agent that reduces albuminuria in a rat model of progressive diabetic nephropathy. Bioorg. Med. Chem. Lett. 2013, 23, 6868– 6873, DOI: 10.1016/j.bmcl.2013.09.100[Crossref], [PubMed], [CAS], Google Scholar241https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1yqurrN&md5=2f74b5facaf9c7c186e18ce7bac5dfbb3',4'-Bis-difluoromethoxycinnamoylanthranilate (FT061): An orally-active antifibrotic agent that reduces albuminuria in a rat model of progressive diabetic nephropathyWilliams, Spencer J.; Zammit, Steven C.; Cox, Alison J.; Shackleford, David M.; Morizzi, Julia; Zhang, Yuan; Powell, Andrew K.; Gilbert, Richard E.; Krum, Henry; Kelly, Darren J.Bioorganic & Medicinal Chemistry Letters (2013), 23 (24), 6868-6873CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Cinnamoylanthranilates including tranilast have been identified as promising antifibrotics that can reduce fibrosis occurring in the kidney during diabetes, thereby delaying and/or preventing kidney dysfunction. Structure-activity relationships aimed at improving potency and metabolic stability have led to the discovery of FT061. This compd., which bears a bis-difluoromethoxy catechol, attenuates TGF-β-stimulated prodn. of collagen in cultured renal mesangial cells (approx 50% at 3 μM). When dosed orally at 20 mg/kg to male Sprague Dawley rats, FT061 exhibited a high bioavailability (73%), Cmax of 200 μM and Tmax of 150 min, and a half-life of 5.4 h. FT061 reduced albuminuria when orally dosed in rats at 200 mg kg/day in a late intervention study of a rat model of progressive diabetic nephropathy. The prepn. of cinnamoylanthranilates is also discussed.242Matsson, E. M.; Palm, J. E.; Eriksson, U. G.; Bottner, P.; Lundahl, A.; Knutson, L.; Lennernas, H. Effects of ketoconazole on the in vivo biotransformation and hepatobiliary transport of the thrombin inhibitor AZD0837 in pigs. Drug Metab. Dispos. 2011, 39, 239– 246, DOI: 10.1124/dmd.110.035022[Crossref], [PubMed], [CAS], Google Scholar242https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhslKmsbs%253D&md5=8e320402263368d92309c69a0a20066bEffects of ketoconazole on the in vivo biotransformation and hepatobiliary transport of the thrombin inhibitor AZD0837 in pigsMatsson, Elin M.; Palm, Johan E.; Eriksson, Ulf G.; Bottner, Pernilla; Lundahl, Anna; Knutson, Lars; Lennernaes, HansDrug Metabolism and Disposition (2011), 39 (2), 239-246CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)Ketoconazole has been shown in clin. trials to increase the plasma exposure of the oral anticoagulant prodrug AZD0837 [(2S)-N-{4-[(Z)-amino(methoxyimino)methyl]benzyl}-1-{(2R)-2-[3-chloro-5-(difluoromethoxy)phenyl]-2-hydroxyethanoyl}-azetidine-2-carboxamide] and its active metabolite, AR-H067637 [(2S)-N-{4-[amino(imino)methyl]benzyl}-1-{(2R)-2-[3-chloro-5-(difluoromethoxy)phenyl]-2-hydroxyethanoyl}-azetidine-2-carboxamide]. To investigate the biotransformation of AZD0837 and the effect of ketoconazole on this process, we used an exptl. model in pigs that allows repeated sampling from three blood vessels, the bile duct, and a perfused intestinal segment. The pigs received AZD0837 (500 mg) given enterally either alone (n = 5) or together with single-dose ketoconazole (600 mg) (n = 6). The prodrug (n = 2) and its active metabolite (n = 2) were also administered i.v. to provide ref. doses. The plasma data revealed considerable interindividual variation in the exposure of the prodrug, intermediate metabolite, and active metabolite. However, AR-H067637 was detected at very high concns. in the bile with low variability (Aebile = 53 ± 6% of the enteral dose), showing that the compd. had indeed been formed in all of the animals and efficiently transported into the bile canaliculi. Concomitant dosing with ketoconazole increased the area under the plasma concn.-time curve for AZD0837 (by 99%) and for AR-H067637 (by 51%). The effect on the prodrug most likely arose from inhibited CYP3A-mediated metab. Reduced metab. also seemed to explain the increased plasma exposure of the active compd. because ketoconazole prolonged the terminal half-life with no apparent effect on the extensive biliary excretion and biliary clearance. These in vivo results were supported by in vitro depletion expts. for AR-H067637 in pig liver microsomes with and without the addn. of ketoconazole.243Zhong, D.; Xie, Z.; Chen, X. Metabolism of pantoprazole involving conjugation with glutathione in rats. J. Pharm. Pharmacol. 2005, 57, 341– 349, DOI: 10.1211/0022357055669[Crossref], [PubMed], [CAS], Google Scholar243https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXisFSqurY%253D&md5=4f8e36d0ce177ef9f18a15195b1fe0c5Metabolism of pantoprazole involving conjugation with glutathione in ratsZhong, Dafang; Xie, Zhiyong; Chen, XiaoyanJournal of Pharmacy and Pharmacology (2005), 57 (3), 341-349CODEN: JPPMAB; ISSN:0022-3573. (Pharmaceutical Press)We have investigated the metab. of pantoprazole and have provided an explanation for the formation mechanism of its metabolites. Metabolites found in the urine of rats after oral administration of pantoprazole sodium (25 mg kg-1) were analyzed by liq. chromatog./ion trap mass spectrometry (LC/MSn). The N-acetylcysteine derivs. of benzimidazole (M1) and pyridine (M2), four pyridine-related metabolites (M3-M6), and three benzimidazole-related metabolites (M7-M9) were found, none of which had been reported previously. Five of the metabolites (M1, M2, M3, M7, and M8) were isolated from the urine of rats after oral administration of pantoprazole sodium by semi-preparative HPLC. Structures of these metabolites were identified by a combination anal. of LC/MSn and 1HNMR spectra. Structures of the remaining four metabolites (M4, M5, M6, and M9) were tentatively assigned through LC/MSn. The metabolites M2, M3, M4, M5 and M6 and the other metabolites (M1, M7, M8, and M9) reflected the fate of the pyridine moiety and the benzimidazole moiety, resp. The proposed formation route of M3-M6 was via initial redn. to mercaptopyridine followed by S-methylation, O-demethylation, and S-oxidn. to the corresponding sulfoxide or sulfone. Meanwhile, M8 and M9 were formed via initial redn. to the 5-difluoromethoxy-1H-benzoimidazole-2-thiol (M7) followed by hydroxylation and S-methylation. The metab. of pantoprazole included an attack by glutathione on the benzimidazole-2-carbon and pyridine-7'-carbon. It is an important metabolic pathway of pantoprazole in rats.244Cenacchi, V.; Battaglia, R.; Cinato, F.; Riccardi, B.; Spinabelli, D.; Brogin, G.; Puccini, P.; Pezzetta, D. In vitro and in vivo metabolism of CHF 6001, a selective phosphodiesterase (PDE4) inhibitor. Xenobiotica 2015, 45, 693– 710, DOI: 10.3109/00498254.2015.1014945[Crossref], [PubMed], [CAS], Google Scholar244https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFKhtrbN&md5=800594cece11d6c6f7a830d701183a45In vitro and in vivo metabolism of CHF 6001, a selective phosphodiesterase (PDE4) inhibitorCenacchi, Valentina; Battaglia, Rosangela; Cinato, Flavio; Riccardi, Benedetta; Spinabelli, Daniele; Brogin, Giandomenico; Puccini, Paola; Pezzetta, DanieleXenobiotica (2015), 45 (8), 693-710CODEN: XENOBH; ISSN:0049-8254. (Informa Healthcare)1. The metab. of CHF 6001, a novel PDE4 inhibitor, was detd. in vitro in mouse, rat, dog, monkey and human microsomes and hepatocytes and in vivo in plasma, urine, feces and bile of rats after i.v. and intratracheal administration. 2. The behavior of CHF 6001 in microsomes and hepatocytes changed across species. CYP3A4/5 isoenzymes were identified to be the primary enzymes responsible for the metab. of CHF 6001 in human liver microsomes. 3. In the rat, CHF 6001 was found extensively metabolized in urine, feces and bile, but not in plasma, where CHF 6001 was the main compd. present. The metabolite profiles were different in the four biol. matrixes from both qual. and quant. point of view. 4. CHF 6001 was metabolized through hydrolysis with the formation of the alc. CHF 5956, loss of a chlorine atom, loss of the N-oxide, hydroxylation, loss of the cyclopropylmethyl group in the alc. moiety, conjugation with glucuronic acid, glutathione and cysteine-glycine. 5. The major metabolite present in the bile was isolated and characterized by NMR anal. It derived from CHF 6001 through contraction of the pyridine-N-oxide ring to N-hydroxy pyrrole and conjugation with glucuronic acid.245Boland, S.; Alen, J.; Bourin, A.; Castermans, K.; Boumans, N.; Panitti, L.; Vanormelingen, J.; Leysen, D.; Defert, O. Novel Roflumilast analogs as soft PDE4 inhibitors. Bioorg. Med. Chem. Lett. 2014, 24, 4594– 4597, DOI: 10.1016/j.bmcl.2014.07.016[Crossref], [PubMed], [CAS], Google Scholar245https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVWqs7jM&md5=941e29eda1ba0bb3b2da0bbe14383738Novel Roflumilast analogs as soft PDE4 inhibitorsBoland, Sandro; Alen, Jo; Bourin, Arnaud; Castermans, Karolien; Boumans, Nicki; Panitti, Laura; Vanormelingen, Jessica; Leysen, Dirk; Defert, OlivierBioorganic & Medicinal Chemistry Letters (2014), 24 (18), 4594-4597CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)PDE4 inhibitors are of high interest for treatment of a wide range of inflammatory or autoimmune diseases. Their potential however has not yet been realized due to target-assocd. side effects, resulting in a low therapeutic window. We herein report the design, synthesis and evaluation of novel PDE4 inhibitors contg. a γ-lactone structure. Such mols. are designed to undergo metabolic inactivation when entering circulation, thereby limiting systemic exposure and reducing the risk for side effects. The resulting inhibitors, e.g., I (n = 3, 4), were highly active on both PDE4B1 and PDE4D2 and underwent rapid degrdn. in human plasma by paraoxonase 1. In contrast, their metabolites displayed markedly reduced permeability and/or on-target activity.246Moretto, N.; Caruso, P.; Bosco, R.; Marchini, G.; Pastore, F.; Armani, E.; Amari, G.; Rizzi, A.; Ghidini, E.; De Fanti, R.; Capaldi, C.; Carzaniga, L.; Hirsch, E.; Buccellati, C.; Sala, A.; Carnini, C.; Patacchini, R.; Delcanale, M.; Civelli, M.; Villetti, G.; Facchinetti, F. CHF6001 I: a novel highly potent and selective phosphodiesterase 4 inhibitor with robust anti-inflammatory activity and suitable for topical pulmonary administration. J. Pharmacol. Exp. Ther. 2015, 352, 559– 567, DOI: 10.1124/jpet.114.220541[Crossref], [PubMed], [CAS], Google Scholar246https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXit1Omurw%253D&md5=2be98d624fe49f7aba8673fa8aea8688CHF6001 I: a novel highly potent and selective phosphodiesterase 4 inhibitor with robust anti-inflammatory activity and suitable for topical pulmonary administrationMoretto, Nadia; Caruso, Paola; Bosco, Raffaella; Marchini, Gessica; Pastore, Fiorella; Armani, Elisabetta; Amari, Gabriele; Rizzi, Andrea; Ghidini, Eleonora; De Fanti, Renato; Capaldi, Carmelida; Carzaniga, Laura; Hirsch, Emilio; Buccellati, Carola; Sala, Angelo; Carnini, Chiara; Patacchini, Riccardo; Delcanale, Maurizio; Civelli, Maurizio; Villetti, Gino; Facchinetti, FabrizioJournal of Pharmacology and Experimental Therapeutics (2015), 352 (3), 559-567, 9 pp.CODEN: JPETAB; ISSN:1521-0103. (American Society for Pharmacology and Experimental Therapeutics)This study examd. the pharmacol. characterization of CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide], a novel phosphodiesterase (PDE)4 inhibitor designed for treating pulmonary inflammatory diseases via inhaled administration. CHF6001 was 7- and 923-fold more potent than roflumilast and cilomilast, resp., in inhibiting PDE4 enzymic activity (IC50 = 0.026 ± 0.006 nM). CHF6001 inhibited PDE4 isoforms A-D with equal potency, showed an elevated ratio of high-affinity rolipram binding site vs. low-affinity rolipram binding site (i.e., >40) and displayed >20,000-fold selectivity vs. PDE4 compared with a panel of PDEs. CHF6001 effectively inhibited (subnanomolar IC50 values) the release of tumor necrosis factor-α from human peripheral blood mononuclear cells, human acute monocytic leukemia cell line macrophages (THP-1), and rodent macrophages (RAW264.7 and NR8383). Moreover, CHF6001 potently inhibited the activation of oxidative burst in neutrophils and eosinophils, neutrophil chemotaxis, and the release of interferon-γ from CD4+ T cells. In all these functional assays, CHF6001 was more potent than previously described PDE4 inhibitors, including roflumilast, UK-500,001 [2-(3,4-difluorophenoxy)-5-fluoro-N-((1S,4S)-4-(2-hydroxy-5-methylbenzamido)cyclohexyl)nicotinamide], and cilomilast, and it was comparable to GSK256066 [6-((3-(dimethylcarbamoyl)phenyl)sulfonyl)-4-((3-methoxyphenyl)amino)-8-methylquinoline-3-carboxamide]. When administered intratracheally to rats as a micronized dry powder, CHF6001 inhibited liposaccharide-induced pulmonary neutrophilia (ED50 = 0.205 μmol/kg) and leukocyte infiltration (ED50 = 0.188 μmol/kg) with an efficacy comparable to a high dose of budesonide (1 μmol/kg i.p.). In sum, CHF6001 has the potential to be an effective topical treatment of conditions assocd. with pulmonary inflammation, including asthma and chronic obstructive pulmonary disease.247Zhou, Z. Z.; Ge, B. C.; Zhong, Q. P.; Huang, C.; Cheng, Y. F.; Yang, X. M.; Wang, H. T.; Xu, J. P. Development of highly potent phosphodiesterase 4 inhibitors with anti-neuroinflammation potential: Design, synthesis, and structure-activity relationship study of catecholamides bearing aromatic rings. Eur. J. Med. Chem. 2016, 124, 372– 379, DOI: 10.1016/j.ejmech.2016.08.052[Crossref], [PubMed], [CAS], Google Scholar247https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFWisLzL&md5=a94bd083cfeb43381366a854a1624516Development of highly potent phosphodiesterase 4 inhibitors with anti-neuroinflammation potential: Design, synthesis, and structure-activity relationship study of catecholamides bearing aromatic ringsZhou, Zhong-Zhen; Ge, Bing-Chen; Zhong, Qiu-Ping; Huang, Chang; Cheng, Yu-Fang; Yang, Xue-Mei; Wang, Hai-Tao; Xu, Jiang-PingEuropean Journal of Medicinal Chemistry (2016), 124 (), 372-379CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)In this study, catecholamides I (R=Me,CHF2;R1=cyclopentyl, cyclopropylmethyl; R2=H,Me; R3=H,Cl;X=Y=N,C) bearing different arom. rings (such as pyridin-2-yl, pyridin-3-yl,Ph,and 2-chlorophenyl groups) were synthesized as potent phosphodiesterase(PDE)4 inhibitors. The inhibitory activities of these compds. were evaluated against the core catalytic domains of human PDE4(PDE4CAT), full-length PDE4A4, PDE4B1, PDE4C1, and PDE4D7 enzymes, and other PDE family members. Eight of the synthesized compds. were identified as having submicromolar IC50 values in the mid-to low-nanomolar range. Careful anal. on the structure-activity relationship of compds. I revealed that the replacement of the 4-methoxy group with the difluoromethoxy group improved inhibitory activities. More interesting, 4-difluoromethoxybenzamides I (R=CHF2; R1=cyclopentyl, cyclopropylmethyl; R2=H; R3=Cl; X=Y=C) exhibited preference for PDE4 with higher selectivities of about 3333 and 1111-fold over other PDEs, resp. In addn., compd. I (R=CHF2; R1=cyclopropylmethyl; R2=H; R3=Cl; X=Y=C) with wonderful PDE4D7 inhibitory activities inhibited LPS-induced TNF-α prodn. in microglia.248Brullo, C.; Massa, M.; Villa, C.; Ricciarelli, R.; Rivera, D.; Pronzato, M. A.; Fedele, E.; Barocelli, E.; Bertoni, S.; Flammini, L.; Bruno, O. Synthesis, biological activities and pharmacokinetic properties of new fluorinated derivatives of selective PDE4D inhibitors. Bioorg. Med. Chem. 2015, 23, 3426– 3435, DOI: 10.1016/j.bmc.2015.04.027[Crossref], [PubMed], [CAS], Google Scholar248https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmslGisL0%253D&md5=be893b649c0211a01e2b4a15fa14a999Synthesis, biological activities and pharmacokinetic properties of new fluorinated derivatives of selective PDE4D inhibitorsBrullo, Chiara; Massa, Matteo; Villa, Carla; Ricciarelli, Roberta; Rivera, Daniela; Pronzato, Maria Adelaide; Fedele, Ernesto; Barocelli, Elisabetta; Bertoni, Simona; Flammini, Lisa; Bruno, OlgaBioorganic & Medicinal Chemistry (2015), 23 (13), 3426-3435CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)A new series of selective PDE4D inhibitors has been designed and synthesized by replacing 3-methoxy group with 3-difluoromethoxy isoster moiety in our previously reported cathecolic structures. All compds. showed a good PDE4D3 inhibitory activity, most of them being inactive toward other PDE4 isoforms (PDE4A4, PDE4B2 and PDE4C2). Compd. 3b, chosen among the synthesized compds. as the most promising in terms of inhibitory activity, selectivity and safety, showed an improved pharmacokinetic profile compared to its non fluorinated analog. Spontaneous locomotor activity, assessed in an open field app., showed that, differently from rolipram and diazepam, selective PDE4D inhibitors, such as compds. 3b, 5b and 7b, did not affect locomotion, whereas compd. 1b showed a tendency to reduce the distance traveled and to prolong the immobility period, possibly due to a poor selectivity.249Guay, D.; Hamel, P.; Blouin, M.; Brideau, C.; Chan, C. C.; Chauret, N.; Ducharme, Y.; Huang, Z.; Girard, M.; Jones, T. R.; Laliberte, F.; Masson, P.; McAuliffe, M.; Piechuta, H.; Silva, J.; Young, R. N.; Girard, Y. Discovery of L-791,943: a potent, selective, non emetic and orally active phosphodiesterase-4 inhibitor. Bioorg. Med. Chem. Lett. 2002, 12, 1457– 1461, DOI: 10.1016/S0960-894X(02)00190-7[Crossref], [PubMed], [CAS], Google Scholar249https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xjsl2lsbY%253D&md5=d52b627cf615fa8a862625ae001c8181Discovery of L-791,943: A potent, selective, non emetic and orally active phosphodiesterase-4 inhibitorGuay, Daniel; Hamel, Pierre; Blouin, Marc; Brideau, Christine; Chan, Chi Chung; Chauret, Nathalie; Ducharme, Yves; Huang, Zheng; Girard, Mario; Jones, Tom R.; Laliberte, France; Masson, Paul; McAuliffe, Malia; Piechuta, Hanna; Silva, Jose; Young, Robert N.; Girard, YvesBioorganic & Medicinal Chemistry Letters (2002), 12 (11), 1457-1461CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science Ltd.)Structure-activity relationship studies directed toward improving the potency and metabolic stability of CDP-840 resulted in the discovery of L-791,943 as a potent (HWB TNF-α=0.67 μM) and orally active phosphodiesterase type 4 (PDE4) inhibitor. This compd., which bears a stable bis-difluoromethoxy catechol and a pendant hexafluorocarbinol, exhibited a long half-life in rat and in squirrel monkey. It is well tolerated in ferret with an emetic threshold greater than 30 mg/kg (po) and was found to be active in the ovalbumin-induced bronchoconstriction model in guinea pig and in the ascaris-induced bronchoconstriction models in sheep and squirrel monkey.250Zhuo, X.; Hartz, R. A.; Bronson, J. J.; Wong, H.; Ahuja, V. T.; Vrudhula, V. M.; Leet, J. E.; Huang, S.; Macor, J. E.; Shu, Y. Z. Comparative biotransformation of pyrazinone-containing corticotropin-releasing factor receptor-1 antagonists: minimizing the reactive metabolite formation. Drug Metab. Dispos. 2010, 38, 5– 15, DOI: 10.1124/dmd.109.028910[Crossref], [PubMed], [CAS], Google Scholar250https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhs1aru7fE&md5=ce4d9c5f99f96cbc018968130b068e81Comparative biotransformation of pyrazinone-containing corticotropin-releasing factor receptor-1 antagonists: minimizing the reactive metabolite formationZhuo, Xiaoliang; Hartz, Richard A.; Bronson, Joanne J.; Wong, Harvey; Ahuja, Vijay T.; Vrudhula, Vivekananda M.; Leet, John E.; Huang, Stella; Macor, John E.; Shu, Yue-ZhongDrug Metabolism and Disposition (2010), 38 (1), 5-15CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)(S)-5-Chloro-1-(1-cyclopropylethyl)-3-(2,6-dichloro-4-(trifluoromethyl)-phenylamino)pyrazin-2(1H)-one (BMS-665053), a pyrazinone-contg. compd., is a potent and selective antagonist of corticotropin-releasing factor receptor-1 (CRF-R1) that showed efficacy in the defensive withdrawal model for anxiety in rats, suggesting its use as a potential treatment for anxiety and depression. In vitro metab. studies of BMS-665053 in rat and human liver microsomes revealed cytochrome P 450-mediated oxidn. of the pyrazinone moiety, followed by ring opening, as the primary metabolic pathway. Detection of a series of GSH adducts in trapping expts. suggested the formation of a reactive intermediate, probably as a result of epoxidn. of the pyrazinone moiety. In addn., BMS-665053 (20 mg/kg i.v.) underwent extensive metab. in bile duct-cannulated (BDC) rats. The major drug-related materials in rat plasma were the pyrazinone oxidn. products. In rat bile and urine (0-7 h), only a trace amt. of the parent drug was recovered, whereas significant levels of the pyrazinone epoxide-derived metabolites and GSH-related conjugates were detected. Further evidence suggested that GSH-related conjugates also formed at the dichloroarylamine moiety possibly via an epoxide or a quinone imine intermediate. Other major metabolites in BDC rat bile and urine included glucuronide conjugates. To reduce potential liability due to metabolic activation of BMS-665053, a no. of pyrazinone analogs with different substituents were synthesized and investigated for reactive metabolite formation, leading to the discovery of a CRF-R1 antagonist with diminished in vitro metabolic activation.251Olah, G. A.; Nojima, M.; Kerekes, I. Synthetic methods and reactions. IV. Fluorination of carboxylic acids with cyanuric fluoride. Synthesis 1973, 1973, 487– 488, DOI: 10.1055/s-1973-22238252Wei, C.; Chupak, L. S.; Philip, T.; Johnson, B. M.; Gentles, R.; Drexler, D. M. Screening and characterization of reactive compounds with in vitro peptide-trapping and liquid chromatography/high-resolution accurate mass spectrometry. J. Biomol. Screening 2014, 19, 297– 307, DOI: 10.1177/1087057113492852[Crossref], [PubMed], [CAS], Google Scholar252https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhslWgs7vN&md5=49f09c44ffdd887b3523575a8af4d838Screening and characterization of reactive compounds with in vitro peptide-trapping and liquid chromatography/high-resolution accurate mass spectrometryWei, Cong; Chupak, Louis S.; Philip, Thomas; Johnson, Benjamin M.; Gentles, Robert; Drexler, Dieter M.Journal of Biomolecular Screening (2014), 19 (2), 297-307, 11CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)The present study describes a novel methodol. for the detection of reactive compds. using in vitro peptide trapping and liq. chromatog.-high-resoln. accurate mass spectrometry (LC-HRMS). Compds. that contain electrophilic groups can covalently bind to nucleophilic moieties in proteins and form adducts. Such adducts are thought to be assocd. with drug-mediated toxicity and therefore represent potential liabilities in drug discovery programs. In addn., reactive compds. identified in biol. screening can be assocd. with data that can be misinterpreted if the reactive nature of the compd. is not appreciated. In this work, to facilitate the triage of hits from high-throughput screening (HTS), a novel assay was developed to monitor the formation of covalent peptide adducts by compds. suspected to be chem. reactive. The assay consists of in vitro incubations of test compds. (under conditions of physiol. pH) with synthetically prepd. peptides presenting a variety of nucleophilic moieties such as cysteine, lysine, histidine, arginine, serine, and tyrosine. Reaction mixts. were analyzed using full-scan LC-HRMS, the data were interrogated using postacquisition data mining, and modified amino acids were identified by subsequent LC-HRMS/mass spectrometry. The study demonstrated that in vitro nucleophilic peptide trapping followed by LC-HRMS anal. is a useful approach for screening of intrinsically reactive compds. identified from HTS exercises, which are then removed from follow-up processes, thus obviating the generation of data from biochem. activity assays.253Rodil, A.; Slawin, A. M. Z.; Al-Maharik, N.; Tomita, R.; O’Hagan, D. O. Fluorine-containing substituents: metabolism of the α,α-difluoroethyl thioether motif. Beilstein J. Org. Chem. 2019, 15, 1441– 1447, DOI: 10.3762/bjoc.15.144[Crossref], [PubMed], [CAS], Google Scholar253https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1eitbvJ&md5=967d18a4e9cf6c8f3935a4a7e19065d0Fluorine-containing substituents: metabolism of the α,α-difluoroethyl thioether motifRodil, Andrea; Slawin, Alexandra M. Z.; Al-Maharik, Nawaf; Tomita, Ren; O'Hagan, DavidBeilstein Journal of Organic Chemistry (2019), 15 (), 1441-1447CODEN: BJOCBH; ISSN:1860-5397. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)We report the metab. of the recently introduced α,α-difluoroethyl thioether motif to explore further its potential as a substituent for bioactives discovery chem. Incubation of two aryl-SCF2CH3 ethers with the model yeast organism Cunninghamella elegans, indicates that the sulfur of the thioether is rapidly converted to the corresponding sulfoxide, and then significantly more slowly to the sulfone. When the substrate was (p-OMe)PhSCF2CH3, then the resultant (demethylated) phenol sulfoxide had an enantiomeric excess of 60%, and when the substrate was the β-substituted-SCF2CH3 naphthalene, then the enantiomeric excess of the resultant sulfoxide was 54%. There was no evidence of defluorination, unlike the corresponding oxygen ether (p-OMe)PhOCF2CH3, which was converted to the (demethylated) phenol acetate ester during C. elegans incubation. We conclude that the aryl-S-CF2CH3 motif is metabolised in a similar manner to aryl-SCF3, a motif that is being widely explored in discovery chem. It is however, significantly less lipophilic than aryl-SCF3 which may offer a practical advantage in tuning overall pharmacokinetic profiles of mols. in development.254Tomita, R.; Al-Maharik, N.; Rodil, A.; Buhl, M.; O’Hagan, D. Synthesis of aryl α,α-difluoroethyl thioethers a novel structure motif in organic chemistry, and extending to aryl α,α-difluoro oxyethers. Org. Biomol. Chem. 2018, 16, 1113– 1117, DOI: 10.1039/C7OB02987J[Crossref], [PubMed], [CAS], Google Scholar254https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXosVKmsw%253D%253D&md5=c8144df20378eac6ac827338da2bba94Synthesis of aryl α,α-difluoroethyl thioethers a novel structure motif in organic chemistry, and extending to aryl α,α-difluoro oxyethersTomita, Ren; Al-Maharik, Nawaf; Rodil, Andrea; Buhl, Michael; O'Hagan, DavidOrganic & Biomolecular Chemistry (2018), 16 (7), 1113-1117CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)A method for the prepn. of aryl α,α-difluoroethyl thioethers ArSCF2CH3 (Ar = C6H5, naphthalen-2-yl, 4-(cyclohexyloxy)phenyl, etc.) is reported and the synthesis approach is extended to aryl α,α-difluoroethyl oxygen ethers 4-RC6H4OCF2CH3 (R = H, OCH3, C6H5). Selected building blocks are further elaborated in cross-coupling reactions and are incorporated into analogs of established trifluoromethyl ether drugs. Conformations are explored and log P studies of these motifs indicate that they are significantly more polar than their trifluoromethyl ether analogs rendering them attractive for bioactives discovery.255Murray, M. Mechanisms of inhibitory and regulatory effects of methylenedioxyphenyl compounds on cytochrome P450-dependent drug oxidation. Curr. Drug Metab. 2000, 1, 67– 84, DOI: 10.2174/1389200003339270[Crossref], [PubMed], [CAS], Google Scholar255https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXlt1Whurs%253D&md5=dcafc824c8bcd0080d256d6dd8c50ae7Mechanisms of inhibitory and regulatory effects of methylenedioxyphenyl compounds on cytochrome P450-dependent drug oxidationMurray, MichaelCurrent Drug Metabolism (2000), 1 (1), 67-84CODEN: CDMUBU ISSN:. (Bentham Science Publishers Ltd.)A review with 110 refs. Cytochrome P 450 (CYP) enzymes catalyze the oxidative conversion of drugs and other lipophilic compds. to hydrophilic metabolites. Thus, CYPs play a dominant role in the elimination of drugs from the body. Inhibitory interactions occur when drugs compete for oxidn. by specific CYPs, whereas certain drugs increase the capacity for oxidative biotransformation by inducing the synthesis of new CYPs. Methylenedioxyphenyl (MDP) compds. have been widely employed as com. important pesticide synergists and a no. of derivs. are found in oils and spices. MDP compds. are of considerable toxicol. significance because of their capacity to inhibit and induce CYP enzymes in mammals; some derivs. produce neurotoxic and hepatotoxic effects. Although there are relatively few therapeutic agents of present clin. importance that possess the MDP structural feature, the synthesis and preclin. evaluation of such agents appears to be increasing. In the context of the existing literature surrounding MDP compds. it is noteworthy that these potential drugs also elicit significant modulatory effects on CYP activities in rat and human liver. These developments indicate the importance of understanding the chem. mechanisms by which MDPs interact with CYPs. Thus, the presence of the MDP structure may undermine the potential clin. value of new drugs.256Bartholomaeus, A. Fludioxonil. In World Health Organization Joint Meeting on Pesticide Residues; World Health Organization, 2004; pp 47– 84.257Keith, J. M.; Jones, W. M.; Tichenor, M.; Liu, J.; Seierstad, M.; Palmer, J. A.; Webb, M.; Karbarz, M.; Scott, B. P.; Wilson, S. J.; Luo, L.; Wennerholm, M. L.; Chang, L.; Rizzolio, M.; Rynberg, R.; Chaplan, S. R.; Breitenbucher, J. G. Preclinical characterization of the FAAH inhibitor JNJ-42165279. ACS Med. Chem. Lett. 2015, 6, 1204– 1208, DOI: 10.1021/acsmedchemlett.5b00353[ACS Full Text], [CAS], Google Scholar257https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhslGms73P&md5=fc681414ace29f39906fdd62c204114bPreclinical Characterization of the FAAH Inhibitor JNJ-42165279Keith, John M.; Jones, William M.; Tichenor, Mark; Liu, Jing; Seierstad, Mark; Palmer, James A.; Webb, Michael; Karbarz, Mark; Scott, Brian P.; Wilson, Sandy J.; Luo, Lin; Wennerholm, Michelle L.; Chang, Leon; Rizzolio, Michele; Rynberg, Raymond; Chaplan, Sandra R.; Breitenbucher, J. GuyACS Medicinal Chemistry Letters (2015), 6 (12), 1204-1208CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)The preclin. characterization of the aryl piperazinyl urea inhibitor of fatty acid amide hydrolase (FAAH) JNJ-42165279 I is described. JNJ-42165279 covalently inactivates the FAAH enzyme, but is highly selective with regard to other enzymes, ion channels, transporters, and receptors. JNJ-42165279 exhibited excellent ADME and pharmacodynamic properties as evidenced by its ability to block FAAH in the brain and periphery of rats and thereby cause an elevation of the concns. of anandamide (AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The compd. was also efficacious in the spinal nerve ligation (SNL) model of neuropathic pain. The combination of good phys., ADME, and PD properties of JNJ-42165279 supported it entering the clin. portfolio.258Rose, W. C.; Marathe, P. H.; Jang, G. R.; Monticello, T. M.; Balasubramanian, B. N.; Long, B.; Fairchild, C. R.; Wall, M. E.; Wani, M. C. Novel fluoro-substituted camptothecins: in vivo antitumor activity, reduced gastrointestinal toxicity and pharmacokinetic characterization. Cancer Chemother. Pharmacol. 2006, 58, 73– 85, DOI: 10.1007/s00280-005-0128-y[Crossref], [PubMed], [CAS], Google Scholar258https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjslGjsbg%253D&md5=97ecbd7d19b5e051255c5c0dc03b215aNovel fluoro-substituted camptothecins: in vivo antitumor activity, reduced gastrointestinal toxicity and pharmacokinetic characterizationRose, William C.; Marathe, Punit H.; Jang, Graham R.; Monticello, Thomas M.; Balasubramanian, Balu N.; Long, Byron; Fairchild, Craig R.; Wall, Monroe E.; Wani, Mansukh C.Cancer Chemotherapy and Pharmacology (2006), 58 (1), 73-85CODEN: CCPHDZ; ISSN:0344-5704. (Springer)Purpose: The novel fluoro-substituted camptothecin analog, BMS-286309, and its prodrug, BMS-422461, were evaluated for their pharmacol., toxicol., metabolic and pharmacokinetic developmental potential. Methods: In vitro and in vivo assays were used to assess the compds. for topoisomerase I activity, antitumor activity, gastrointestinal (GI) toxicity, and pharmacokinetic parameters. Results: BMS-286309-induced topoisomerase I-mediated DNA breaks in vitro and was similar in potency to camptothecin. Both BMS-286309 and -422461 were comparable to irinotecan regarding preclin. antitumor activity assessed in mice bearing distal site murine and human tumors. BMS-422461 was also found to be orally active. Both analogs were >100-fold more potent in vivo than irinotecan and both were superior to irinotecan with respect to toxicol. assessment of GI injury in mice. The generation of parent compd. from BMS-422461 was qual. similar in mouse, rat and human blood and liver S9 fractions. The percentage of BMS-286309 remaining as the active lactone form at equil. was comparable in mouse and human plasma. The pharmacokinetic profile in rat blood demonstrated that BMS-422461 was rapidly cleaved to BMS-286309. Conclusions: The favorable in vivo metabolic activation of BMS-422461, and the pharmacokinetic characteristics of BMS-286309, suggest that the good efficacy of BMS-422461 is derived from robust in vivo release of BMS-286309 in rodents and the likelihood that this biotransformation will be preserved in humans. The comparable antitumor activity of BMS-422461 to irinotecan, as well as reduced preclin. GI toxicity, make this novel camptothecin analog attractive for clin. development.259Perfetti, X.; O’Mathuna, B.; Pizarro, N.; Cuyas, E.; Khymenets, O.; Almeida, B.; Pellegrini, M.; Pichini, S.; Lau, S. S.; Monks, T. J.; Farre, M.; Pascual, J. A.; Joglar, J.; de la Torre, R. Neurotoxic thioether adducts of 3,4-methylenedioxymethamphetamine identified in human urine after ecstasy ingestion. Drug Metab. Dispos. 2009, 37, 1448– 1455, DOI: 10.1124/dmd.108.026393[Crossref], [PubMed], [CAS], Google Scholar259https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXosFemsrs%253D&md5=e32e277a1b10c6423f624afecca89f6fNeurotoxic thioether adducts of 3,4-methylenedioxymethamphetamine identified in human urine after ecstasy ingestionPerfetti, Ximena; O'Mathuna, Brian; Pizarro, Nieves; Cuyas, Elisabet; Khymenets, Olha; Almeida, Bruno; Pellegrini, Manuela; Pichini, Simona; Lau, Serrine S.; Monks, Terrence J.; Farre, Magi; Pascual, Jose Antonio; Joglar, Jesus; de la Torre, RafaelDrug Metabolism and Disposition (2009), 37 (7), 1448-1455CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) is a widely misused synthetic amphetamine deriv. and a serotonergic neurotoxicant in animal models and possibly humans. The underlying mechanism of neurotoxicity involves the formation of reactive oxygen species although their source remains unclear. It has been postulated that MDMA-induced neurotoxicity is mediated via the formation of bioreactive metabolites. In particular, the primary catechol metabolites, 3,4-dihydroxymethamphetamine (HHMA) and 3,4-dihydroxyamphetamine (HHA), subsequently cause the formation of glutathione and N-acetylcysteine conjugates, which retain the ability to redox cycle and are serotonergic neurotoxicants in rats. Although the presence of such metabolites has been recently demonstrated in rat brain microdialyzate, their formation in humans has not been reported. The present study describes the detection of 5-(N-acetylcystein-S-yl)-3,4-dihydroxymethamphetamine (N-Ac-5-Cys-HHMA) and 5-(N-acetylcystein-S-yl)-3,4-dihydroxyamphetamine (N-Ac-5-Cys-HHA) in human urine of 15 recreational users of MDMA (1.5 mg/kg) in a controlled setting. The results reveal that in the first 4 h after MDMA ingestion ∼0.002% of the administered dose was recovered as thioether adducts. Genetic polymorphisms in CYP2D6 and catechol-O-methyltransferase expression, the combination of which are major determinants of steady-state levels of HHMA and 4-hydroxy-3-methoxyamphetamine, probably explain the interindividual variability seen in the recovery of N-Ac-5-Cys-HHMA and N-Ac-5-Cys-HHA. In summary, the formation of neurotoxic thioether adducts of MDMA has been demonstrated for the first time in humans. The findings lend wt. to the hypothesis that the bioactivation of MDMA to neurotoxic metabolites is a relevant pathway to neurotoxicity in humans.260Ot’alora G, M.; Grigsby, J.; Poulter, B.; Van Derveer, J. W., III; Giron, S. G.; Jerome, L.; Feduccia, A. A.; Hamilton, S.; Yazar-Klosinski, B.; Emerson, A.; Mithoefer, M. C.; Doblin, R. 3,4-Methylenedioxymethamphetamine-assisted psychotherapy for treatment of chronic posttraumatic stress disorder: A randomized phase 2 controlled trial. J. Psychopharmacol. 2018, 32, 1295– 1307, DOI: 10.1177/0269881118806297[Crossref], [PubMed], [CAS], Google Scholar260https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXit1ymur%252FL&md5=a3cff037100d9034378f62498b4e7eba3,4-Methylenedioxymethamphetamine-assisted psychotherapy for treatment of chronic posttraumatic stress disorder: A randomized phase 2 controlled trialOt'alora G, Marcela; Grigsby, Jim; Poulter, Bruce; Van Derveer, Joseph W.; Giron, Sara Gael; Jerome, Lisa; Feduccia, Allison A.; Hamilton, Scott; Yazar-Klosinski, Berra; Emerson, Amy; Mithoefer, Michael C.; Doblin, RickJournal of Psychopharmacology (London, United Kingdom) (2018), 32 (12), 1295-1307CODEN: JOPSEQ; ISSN:0269-8811. (Sage Publications Ltd.)Posttraumatic stress disorder often does not resolve after conventional psychotherapies or pharmacotherapies. Pilot studies have reported that 3,4-methylenedioxymethamphetamine (MDMA) combined with psychotherapy reduces posttraumatic stress disorder symptoms. This pilot dose response trial assessed efficacy and safety of MDMA-assisted psychotherapy across multiple therapy teams. Twenty-eight people with chronic posttraumatic stress disorder were randomized in a double-blind dose response comparison of two active doses (100 and 125 mg) with a low dose (40 mg) of MDMA administered during eight-hour psychotherapy sessions. Change in the Clinician-Administered PTSD Scale total scores one month after two sessions of MDMA served as the primary outcome. Active dose groups had one addnl. open-label session; the low dose group crossed over for three open-label active dose sessions. A 12-mo follow-up assessment occurred after the final MDMA session. In the intent-to-treat set, the active groups had the largest redn. in Clinician-Administered PTSD Scale total scores at the primary endpoint, with mean (std. deviation) changes of -26.3 (29.5) for 125 mg, -24.4 (24.2) for 100 mg, and -11.5 (21.2) for 40 mg, though statistical significance was reached only in the per protocol set (p = 0.03). Posttraumatic stress disorder symptoms remained lower than baseline at 12-mo follow-up (p<0.001) with 76% (n = 25) not meeting posttraumatic stress disorder criteria. There were no drug-related serious adverse events, and the treatment was well-tolerated. Our findings support previous investigations of MDMA-assisted psychotherapy as an innovative, efficacious treatment for posttraumatic stress disorder.261Ricaurte, G. A.; Yuan, J.; Hatzidimitriou, G.; Cord, B. J.; McCann, U. D. Retraction. Science 2003, 301, 1479, DOI: 10.1126/science.301.5639.1479b[Crossref], [PubMed], [CAS], Google Scholar261https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXnt1Kqtr4%253D&md5=2d714a34872298db242f49dd3003c49cRetractionRicaurte, George A.; Yuan, Jie; Hatzidmitriou, George; Cord, Branden J.; McCann, Una D.Science (Washington, DC, United States) (2003), 301 (5639), 1479CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)There is no expanded citation for this reference.262Center for Drug Evaluation and Research. Application Number: 206038Orig1s000. Clinical Pharmacology and Biopharmaceutics Review(s). https://www.accessdata.fda.gov/drugsatfda_docs/nda/2015/0206038Orig1s000ClinPharmR.pdf (accessed 2019-05-06).263Bertelsen, K. M.; Venkatakrishnan, K.; Von Moltke, L. L.; Obach, R. S.; Greenblatt, D. J. Apparent mechanism-based inhibition of human CYP2D6 in vitro by paroxetine: comparison with fluoxetine and quinidine. Drug Metab. Dispos. 2003, 31, 289– 293, DOI: 10.1124/dmd.31.3.289[Crossref], [PubMed], [CAS], Google Scholar263https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhsFKlt7Y%253D&md5=2336fbc058e49e7b530562809c62ba09Apparent mechanism-based inhibition of human CYP2D6 in vitro by paroxetine: Comparison with fluoxetine and quinidineBertelsen, Kirk M.; Venkatakrishnan, Karthik; Von Moltke, Lisa L.; Obach, R. Scott; Greenblatt, David J.Drug Metabolism and Disposition (2003), 31 (3), 289-293CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)Paroxetine, a selective serotonin reuptake inhibitor, is a potent inhibitor of cytochrome P 450 2D6 (CYP2D6) activity, but the mechanism of inhibition is not established. To det. whether preincubation affects the inhibition of human liver microsomal dextromethorphan demethylation activity by paroxetine, we used a two-step incubation scheme in which all of the enzyme assay components, minus substrate, are preincubated with paroxetine. The kinetic parameters of inhibition were also estd. by varying the time of preincubation as well as the concn. of inhibitor. From these data, a Kitz-Wilson plot was constructed, allowing the estn. of both an apparent inactivator concn. required for half-maximal inactivation (KI) and the maximal rate const. of inactivation (KINACT) value for this interaction. Preincubation of paroxetine with human liver microsomes caused an approx. 8-fold redn. in the IC50 value (0.34 vs. 2.54 μM). Time-dependent inhibition was demonstrated with an apparent KI of 4.85 μM and an apparent KINACT value of 0.17 min-1. Spectral scanning of CYP2D6 with paroxetine yielded an increase in absorbance at 456 nm suggesting paroxetine inactivation of CYP2D6 via the formation of a metabolite intermediate complex. This pattern is consistent with the metab. of the methylenedioxy substituent in paroxetine; such substituents may produce mechanism-based inactivation of cytochrome P 450 enzymes. In contrast, quinidine and fluoxetine, both of which are inhibitors of CYP2D6 activity, did not exhibit a preincubation-dependent increase in inhibitory potency. These data are consistent with mechanism-based inhibition of CYP2D6 by paroxetine but not by quinidine or fluoxetine.264Halazy, S.; Danzin, C.; Ehrhard, A.; Gerhart, F. 1,1-Difluoroalkyl glucosides: a new class of enzyme-activated irreversible inhibitors of α-glucosidases. J. Am. Chem. Soc. 1989, 111, 3484– 3485, DOI: 10.1021/ja00191a085[ACS Full Text], [CAS], Google Scholar264https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXhvFehtrs%253D&md5=8f71ee79807e7ee4cd2713e3c338ce681,1-Difluoroalkyl glucosides: a new class of enzyme-activated irreversible inhibitors of α-glucosidasesHalazy, S.; Danzin, C.; Ehrhard, A.; Gerhart, F.Journal of the American Chemical Society (1989), 111 (9), 3484-5CODEN: JACSAT; ISSN:0002-7863.2'-Chloro-1',1',2'-trifluoroethyl α-D-glucopyranoside (I) was synthesized as a potential inhibitor of α-glucosidase. I produced a time-dependent irreversible inhibition of yeast α-glucosidase. The inactivation is explained by the enzyme-catalyzed hydrolysis of the glycosidic linkage releasing a fluorohydrin which rapidly decomps. to an acyl fluoride and acetylates a nucleophilic residue in the active site. I was found to be a substrate of the sucrase-isomaltase complex purified from rat small intestine.265Vocadlo, D. J.; Davies, G. J. Mechanistic insights into glycosidase chemistry. Curr. Opin. Chem. Biol. 2008, 12, 539– 555, DOI: 10.1016/j.cbpa.2008.05.010[Crossref], [PubMed], [CAS], Google Scholar265https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXht1KgsbnF&md5=dc269026d21f0e114c25b120b3c1e9b9Mechanistic insights into glycosidase chemistryVocadlo, David J.; Davies, Gideon J.Current Opinion in Chemical Biology (2008), 12 (5), 539-555CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. The enzymic hydrolysis of the glycosidic bond continues to gain importance, reflecting the critically important roles complex glycans play in health and disease as well as the rekindled interest in enzymic biomass conversion. Recent advances include the broadening of our understanding of enzyme reaction coordinates, through both computational and structural studies, improved understanding of enzyme inhibition through transition state mimicry and fascinating insights into mechanism yielded by phys. org. chem. approaches.266Spahn, V.; Del Vecchio, G.; Labuz, D.; Rodriguez-Gaztelumendi, A.; Massaly, N.; Temp, J.; Durmaz, V.; Sabri, P.; Reidelbach, M.; Machelska, H.; Weber, M.; Stein, C. A nontoxic pain killer designed by modeling of pathological receptor conformations. Science 2017, 355, 966– 969, DOI: 10.1126/science.aai8636[Crossref], [PubMed], [CAS], Google Scholar266https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjsVCgs7k%253D&md5=719809495aec8c3ac9e60429c9edea98A nontoxic pain killer designed by modeling of pathological receptor conformationsSpahn, V.; Del Vecchio, G.; Labuz, D.; Rodriguez-Gaztelumendi, A.; Massaly, N.; Temp, J.; Durmaz, V.; Sabri, P.; Reidelbach, M.; Machelska, H.; Weber, M.; Stein, C.Science (Washington, DC, United States) (2017), 355 (6328), 966-969CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Indiscriminate activation of opioid receptors provides pain relief but also severe central and intestinal side effects. We hypothesized that exploiting pathol. (rather than physiol.) conformation dynamics of opioid receptor-ligand interactions might yield ligands without adverse actions. By computer simulations at low pH, a hallmark of injured tissue, we designed an agonist that, because of its low acid dissocn. const., selectively activates peripheral μ-opioid receptors at the source of pain generation. Unlike the conventional opioid fentanyl, this agonist showed pH-sensitive binding, heterotrimeric guanine nucleotide-binding protein (G protein) subunit dissocn. by fluorescence resonance energy transfer, and adenosine 3',5'-monophosphate inhibition in vitro. It produced injury-restricted analgesia in rats with different types of inflammatory pain without exhibiting respiratory depression, sedation, constipation, or addiction potential.267Grunewald, G. L.; Seim, M. R.; Lu, J.; Makboul, M.; Criscione, K. R. Application of the Goldilocks effect to the design of potent and selective inhibitors of phenylethanolamine N-methyltransferase: balancing pKa and steric effects in the optimization of 3-methyl-1,2,3,4-tetrahydroisoquinoline inhibitors by β-fluorination. J. Med. Chem. 2006, 49, 2939– 2952, DOI: 10.1021/jm051262k[ACS Full Text], [CAS], Google Scholar267https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjsVajtb4%253D&md5=468ae2a5832e69d751628ca743ef951cApplication of the Goldilocks Effect to the Design of Potent and Selective Inhibitors of Phenylethanolamine N-Methyltransferase: Balancing pKa and Steric Effects in the Optimization of 3-Methyl-1,2,3,4-tetrahydroisoquinoline Inhibitors by β-FluorinationGrunewald, Gary L.; Seim, Mitchell R.; Lu, Jian; Makboul, Mariam; Criscione, Kevin R.Journal of Medicinal Chemistry (2006), 49 (10), 2939-2952CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)3-Methyl-1,2,3,4-tetrahydroisoquinolines (3-methyl-THIQs) are potent inhibitors of phenylethanolamine N-methyltransferase (PNMT), but are not selective due to significant affinity for the α2-adrenoceptor. Fluorination of the Me group lowers the pKa of the THIQ amine from 9.53 (CH3) to 7.88 (CH2F), 6.42 (CHF2), and 4.88 (CF3). This decrease in pKa results in a redn. in affinity for the α2-adrenoceptor. However, increased fluorination also results in a redn. in PNMT inhibitory potency, apparently due to steric and electrostatic factors. Biochem. evaluation of a series of 3-fluoromethyl-THIQs and 3-trifluoromethyl-THIQs showed that the former were highly potent inhibitors of PNMT, but were often nonselective due to significant affinity for the α2-adrenoceptor, while the latter were devoid of α2-adrenoceptor affinity, but also lost potency at PNMT. 3-Difluoromethyl-7-substituted-THIQs have the proper balance of both steric and pKa properties and thus have enhanced selectivity vs. the corresponding 3-fluoromethyl-7-substituted-THIQs and enhanced PNMT inhibitory potency vs. the corresponding 3-trifluoromethyl-7-substituted-THIQs. Using the "Goldilocks Effect" analogy, the 3-fluoromethyl-THIQs are too potent (too hot) at the α2-adrenoceptor and the 3-trifluoromethyl-THIQs are not potent enough (too cold) at PNMT, but the 3-difluoromethyl-THIQs are just right. They are both potent inhibitors of PNMT and highly selective due to low affinity for the α2-adrenoceptor. This seems to be the first successful use of the β-fluorination of aliph. amines to impart selectivity to a pharmacol. agent while maintaining potency at the site of interest.268Xu, S.; Zhu, B.; Teffera, Y.; Pan, D. E.; Caldwell, C. G.; Doss, G.; Stearns, R. A.; Evans, D. C.; Beconi, M. G. Metabolic activation of fluoropyrrolidine dipeptidyl peptidase-IV inhibitors by rat liver microsomes. Drug Metab. Dispos. 2005, 33, 121– 130, DOI: 10.1124/dmd.104.001842[Crossref], [PubMed], [CAS], Google Scholar268https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXltlensg%253D%253D&md5=2422da6be675647a96f6b76ab1e88a84Metabolic activation of fluoropyrrolidine dipeptidyl peptidase-IV inhibitors by rat liver microsomesXu, Shiyao; Zhu, Bing; Teffera, Yohannes; Pan, Deborah E.; Caldwell, Charles G.; Doss, George; Stearns, Ralph A.; Evans, David C.; Beconi, Maria G.Drug Metabolism and Disposition (2005), 33 (1), 121-130CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)The current study evaluated the potential for two dipeptidyl peptidase-IV (DPP-IV) inhibitor analogs (1S)-1-(trans-4-{[(4-trifluoromethoxyphenyl)sulfonyl]amino}cyclohexyl)-2-[(3S)-3-fluoropyrrolidin-1-yl]-2-oxoethanaminium chloride and (1S)-1-(trans-4-{[(2,4-difluorophenyl)sulfonyl]amino}cyclohexyl)-2-[(3S)-3-fluoropyrrolidin-1-yl]-2-oxoethanaminium chloride (MRL-A and MRL-B), contg. a fluoropyrrolidine moiety in the structure, to undergo metabolic activation. The irreversible binding of these tritium-labeled compds. to rat liver microsomal protein was time- and NADPH-dependent and was attenuated by the addn. of reduced glutathione (GSH) or N-acetylcysteine (NAC) to the incubation, indicating that chem. reactive intermediates were formed and trapped by these nucleophiles. Mass spectrometric analyses and further trapping expts. with semicarbazide indicated that the fluoropyrrolidine ring had undergone sequential oxidn. and defluorination events resulting in the formation of GSH or NAC conjugates of the pyrrolidine moiety. The bioactivation of MRL-A was catalyzed primarily by rat recombinant CYP3A1 and CYP3A2. Pretreatment of rats with prototypic CYP3A1 and 3A2 inducers (pregnenolone-16α-carbonitrile and dexamethasone) enhanced the extent of bioactivation which, in turn, led to a higher degree of in vitro irreversible binding to microsomal proteins (5- and 9-fold increase, resp.). Herein, the authors describe studies that demonstrate that the fluoropyrrolidine ring is prone to metabolic activation and that GSH or NAC can trap the reactive intermediates to form adducts that provide insight into the mechanisms of bioactivation.269Edmondson, S. D.; Mastracchio, A.; Mathvink, R. J.; He, J.; Harper, B.; Park, Y. J.; Beconi, M.; Di Salvo, J.; Eiermann, G. J.; He, H.; Leiting, B.; Leone, J. F.; Levorse, D. A.; Lyons, K.; Patel, R. A.; Patel, S. B.; Petrov, A.; Scapin, G.; Shang, J.; Roy, R. S.; Smith, A.; Wu, J. K.; Xu, S.; Zhu, B.; Thornberry, N. A.; Weber, A. E. (2S,3S)-3-Amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N-dimethyl-4-oxo-2-(4-[1,2,4]triazolo[1,5-a]-pyridin-6-ylphenyl)butanamide: a selective α-amino amide dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. J. Med. Chem. 2006, 49, 3614– 3627, DOI: 10.1021/jm060015t[ACS Full Text], [CAS], Google Scholar269https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XkslKht7Y%253D&md5=6ebe737acc317676ca1c7e4178d9719f(2S,3S)-3-Amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N-dimethyl-4-oxo-2-(4-[1,2,4]triazolo[1,5-a]- pyridin-6-ylphenyl)butanamide: A selective α-amino amide dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetesEdmondson, Scott D.; Mastracchio, Anthony; Mathvink, Robert J.; He, Jiafang; Harper, Bart; Park, You-Jung; Beconi, Maria; Di Salvo, Jerry; Eiermann, George J.; He, Huaibing; Leiting, Barbara; Leone, Joseph F.; Levorse, Dorothy A.; Lyons, Kathryn; Patel, Reshma A.; Patel, Sangita B.; Petrov, Aleksandr; Scapin, Giovanna; Shang, Jackie; Roy, Ranabir Sinha; Smith, Aaron; Wu, Joseph K.; Xu, Shiyao; Zhu, Bing; Thornberry, Nancy A.; Weber, Ann E.Journal of Medicinal Chemistry (2006), 49 (12), 3614-3627CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of β-substituted biarylphenylalanine amides were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-4) for the treatment of type 2 diabetes. Optimization of the metabolic profile of early analogs led to the discovery of (2S,3S)-3-amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N-dimethyl-4-oxo-2-(4-[1,2,4]triazolo[1,5-a]pyridin-6-ylphenyl)butanamide I, a potent, orally active DPP-4 inhibitor (IC50 = 6.3 nM) with excellent selectivity, oral bioavailability in preclin. species, and in vivo efficacy in animal models. Compd. I was selected for further characterization as a potential new treatment for type 2 diabetes.270Sharma, R.; Sun, H.; Piotrowski, D. W.; Ryder, T. F.; Doran, S. D.; Dai, H.; Prakash, C. Metabolism, excretion, and pharmacokinetics of ((3,3-difluoropyrrolidin-1-yl)((2S,4S)-4-(4-(pyrimidin-2-yl)piperazin-1-yl)pyrrolidin-2-yl)methanone, a dipeptidyl peptidase inhibitor, in rat, dog and human. Drug Metab. Dispos. 2012, 40, 2143– 2161, DOI: 10.1124/dmd.112.047316[Crossref], [PubMed], [CAS], Google Scholar270https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFKkt7rP&md5=8fa2c39deb0ffc3dcda00ee7aa68e250Metabolism, excretion, and pharmacokinetics of ((3,3-difluoropyrrolidin-1-yl)((2S,4S)-4-(4-(pyrimidin-2-yl)piperazin-1-yl)pyrrolidin-2-yl)methanone, a dipeptidyl peptidase inhibitor, in rat, dog and humanSharma, Raman; Sun, Hao; Piotrowski, David W.; Ryder, Tim F.; Doran, Shawn D.; Dai, Haiqing; Prakash, ChandraDrug Metabolism & Disposition (2012), 40 (11), 2143-2161CODEN: DMDSAI; ISSN:1521-009X. (American Society for Pharmacology and Experimental Therapeutics)The disposition of 3,3-difluoropyrrolidin-1-yl{(2S,4S)-4-[4-(pyrimidin-2-yl)piperazin-1-yl]pyrrolidin-2-yl}methanone (PF-00734200), a dipeptidyl peptidase IV inhibitor that progressed to phase 3 for the treatment of type 2 diabetes, was examd. in rats, dogs, and humans after oral administration of a single dose of [14C]PF-00734200. Mean recoveries of administered radioactivity were 97.1, 92.2, and 87.2% in rats, dogs, and humans, resp. The majority of radioactive dose was detected in the urine of dogs and humans and in the feces of rats. Absorption of PF-00734200 was rapid in all species, with maximal plasma concns. of radioactivity achieved within 1 h after the dose. Circulating radioactivity was primarily composed of the parent drug (79.9, 80.2, and 94.4% in rat, dog, and human, resp.). The major route of metab. was due to hydroxylation at the 5' position of the pyrimidine ring (M5) in all species. In vitro expts. with recombinant cytochrome P 450 isoforms suggested that the formation of M5 was catalyzed both by CYP2D6 and CYP3A4. Mol. docking simulations showed that the 5' position of the pyrimidine moiety of PF-00734200 can access the heme iron-oxo of both CYP3A4 and CYP2D6 in an energetically favored orientation. Other metabolic pathways included amide hydrolysis (M2), N-dealkylation at the piperazine nitrogen (M3) and an unusual metabolite resulting from scission of the pyrimidine ring (M1). Phase II metabolic pathways included the following: carbamoyl glucuronidation (M9), glucosidation (M15) on the pyrrolidine nitrogen, and conjugation with creatinine to form an unusual metabolite/metabonate (M16). The data from these studies suggest that PF-00734200 is eliminated by both metab. and renal clearance.271Tremblay, M.; Bethell, R. C.; Cordingley, M. G.; DeRoy, P.; Duan, J.; Duplessis, M.; Edwards, P. J.; Faucher, A. M.; Halmos, T.; James, C. A.; Kuhn, C.; Lacoste, J. E.; Lamorte, L.; LaPlante, S. R.; Malenfant, E.; Minville, J.; Morency, L.; Morin, S.; Rajotte, D.; Salois, P.; Simoneau, B.; Tremblay, S.; Sturino, C. F. Identification of benzofurano[3,2-d]pyrimidin-2-ones, a new series of HIV-1 nucleotide-competing reverse transcriptase inhibitors. Bioorg. Med. Chem. Lett. 2013, 23, 2775– 2780, DOI: 10.1016/j.bmcl.2013.02.042[Crossref], [PubMed], [CAS], Google Scholar271https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXktlCmurk%253D&md5=7dc770482e4deef5332c06b6a31e8a8eIdentification of benzofurano[3,2-d]pyrimidin-2-ones, a new series of HIV-1 nucleotide-competing reverse transcriptase inhibitorsTremblay, Martin; Bethell, Richard C.; Cordingley, Michael G.; DeRoy, Patrick; Duan, Jianmin; Duplessis, Martin; Edwards, Paul J.; Faucher, Anne-Marie; Halmos, Ted; James, Clint A.; Kuhn, Cyrille; Lacoste, Jean-Eric; Lamorte, Louie; LaPlante, Steven R.; Malenfant, Eric; Minville, Joannie; Morency, Louis; Morin, Sebastien; Rajotte, Daniel; Salois, Patrick; Simoneau, Bruno; Tremblay, Sonia; Sturino, Claudio F.Bioorganic & Medicinal Chemistry Letters (2013), 23 (9), 2775-2780CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Screening of our sample collection led to the identification of a set of benzofurano[3,2-d]pyrimidine-2-one hits acting as nucleotide-competing HIV-1 reverse transcriptase inhibitors (NcRTI). Significant improvement in antiviral potency was achieved when substituents were introduced at positions N1, C4, C7 and C8 on the benzofuranopyrimidone scaffold. The series was optimized from low micromolar enzymic activity against HIV-1 RT and no antiviral activity to low nanomolar antiviral potency. Further profiling of inhibitor 30 showed promising overall in vitro properties and also demonstrated that its potency was maintained against viruses resistant to the other major classes of HIV-1 RT inhibitors.272Wang, X.; Sun, M.; New, C.; Nam, S.; Blackaby, W. P.; Hodges, A. J.; Nash, D.; Matteucci, M.; Lyssikatos, J. P.; Fan, P. W.; Tay, S.; Chang, J. H. Probing mechanisms of CYP3A time-dependent inhibition using a truncated model system. ACS Med. Chem. Lett. 2015, 6, 925– 929, DOI: 10.1021/acsmedchemlett.5b00191[ACS Full Text], [CAS], Google Scholar272https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFGitb%252FI&md5=02eb92abebe87a4e406ca9a6241a0471Probing Mechanisms of CYP3A Time-Dependent Inhibition Using a Truncated Model SystemWang, Xiaojing; Sun, Minghua; New, Connie; Nam, Spencer; Blackaby, Wesley P.; Hodges, Alastair J.; Nash, David; Matteucci, Mizio; Lyssikatos, Joseph P.; Fan, Peter W.; Tay, Suzanne; Chang, Jae H.ACS Medicinal Chemistry Letters (2015), 6 (8), 925-929CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)Time-dependent inhibition (TDI) of cytochrome P 450 (CYP) enzymes may incur serious undesirable drug-drug interactions and in rare cases drug-induced idiosyncratic toxicity. The reactive metabolites are often generated through multiple sequential biotransformations and form adducts with CYP enzymes to inactivate their function. The complexity of these processes makes addressing TDI liability very challenging. Strategies to mitigate TDI are therefore highly valuable in discovering safe therapies to benefit patients. In this Letter, the authors disclose the simplified approach toward addressing CYP3A TDI liabilities, guided by metabolic mechanism hypotheses. By adding a Me group onto the α carbon of a basic amine, TDI activities of both the truncated and full mols. I and II were completely eliminated. The authors propose that truncated mols., albeit with caveats, may be used as surrogates for full mols. to investigate TDI.273Wang, X.; Blackaby, W.; Allen, V.; Chan, G. K. Y.; Chang, J. H.; Chiang, P. C.; Diene, C.; Drummond, J.; Do, S.; Fan, E.; Harstad, E. B.; Hodges, A.; Hu, H.; Jia, W.; Kofie, W.; Kolesnikov, A.; Lyssikatos, J. P.; Ly, J.; Matteucci, M.; Moffat, J. G.; Munugalavadla, V.; Murray, J.; Nash, D.; Noland, C. L.; Del Rosario, G.; Ross, L.; Rouse, C.; Sharpe, A.; Slaga, D.; Sun, M.; Tsui, V.; Wallweber, H.; Yu, S. F.; Ebens, A. J. Optimization of pan-Pim kinase activity and oral bioavailability leading to diaminopyrazole (GDC-0339) for the treatment of multiple myeloma. J. Med. Chem. 2019, 62, 2140– 2153, DOI: 10.1021/acs.jmedchem.8b01857[ACS Full Text], [CAS], Google Scholar273https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXisVSqt78%253D&md5=bf8f5e5ac1f4cc29d1b62dcd72e7a815Optimization of Pan-Pim Kinase Activity and Oral Bioavailability Leading to Diaminopyrazole (GDC-0339) for the Treatment of Multiple MyelomaWang, Xiaojing; Blackaby, Wesley; Allen, Vivienne; Chan, Grace Ka Yan; Chang, Jae H.; Chiang, Po-Chang; Diene, Coura; Drummond, Jason; Do, Steven; Fan, Eric; Harstad, Eric B.; Hodges, Alastair; Hu, Huiyong; Jia, Wei; Kofie, William; Kolesnikov, Aleksandr; Lyssikatos, Joseph P.; Ly, Justin; Matteucci, Mizio; Moffat, John G.; Munugalavadla, Veerendra; Murray, Jeremy; Nash, David; Noland, Cameron L.; Del Rosario, Geoff; Ross, Leanne; Rouse, Craig; Sharpe, Andrew; Slaga, Dionysos; Sun, Minghua; Tsui, Vickie; Wallweber, Heidi; Yu, Shang-Fan; Ebens, Allen J.Journal of Medicinal Chemistry (2019), 62 (4), 2140-2153CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Pim kinases have been targets of interest for a no. of therapeutic areas. Evidence of durable single-agent efficacy in human clin. trials validated Pim kinase inhibition as a promising therapeutic approach for multiple myeloma patients. Here, we report the compd. optimization leading to GDC-0339 (16), a potent, orally bioavailable, and well tolerated pan-Pim kinase inhibitor that proved efficacious in RPMI8226 and MM.1S human multiple myeloma xenograft mouse models and has been evaluated as an early development candidate.274Acton, B.; Small, H. F.; Smith, K. M.; McGonagle, A.; Stowell, A. I. J.; James, D. I.; Hamilton, N. M.; Hamilton, N.; Hitchin, J. R.; Hutton, C. P.; Waddell, I. D.; Ogilvie, D. J.; Jordan, A. M. Fluoromethylcyclopropylamine derivatives as potential in vivo toxicophores - A cautionary disclosure. Bioorg. Med. Chem. Lett. 2019, 29, 560– 562, DOI: 10.1016/j.bmcl.2018.12.066[Crossref], [PubMed], [CAS], Google Scholar274https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXkvVegtw%253D%253D&md5=041dbec76f5fbff64ea01e59315cfa2fFluoromethylcyclopropylamine derivatives as potential in vivo toxicophores - A cautionary disclosureActon, Ben; Small, Helen F.; Smith, Kate M.; McGonagle, Alison; Stowell, Alexandra I. J.; James, Dominic I.; Hamilton, Niall M.; Hamilton, Nicola; Hitchin, James R.; Hutton, Colin P.; Waddell, Ian D.; Ogilvie, Donald J.; Jordan, Allan M.Bioorganic & Medicinal Chemistry Letters (2019), 29 (4), 560-562CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Fluorination of metabolic hotspots in a mol. is a common medicinal chem. strategy to improve in vivo half-life and exposure and, generally, this strategy offers significant benefits. Here, we report the application of this strategy to a series of poly-ADP ribose glycohydrolase (PARG) inhibitors, resulting in unexpected in vivo toxicity which was attributed to this single-atom modification.275Berkowitz, D. B.; Karukurichi, K. R.; de la Salud-Bea, R.; Nelson, D. L.; McCune, C. D. Use of fluorinated functionality in enzyme inhibitor development: mechanistic and analytical advantages. J. Fluorine Chem. 2008, 129, 731– 742, DOI: 10.1016/j.jfluchem.2008.05.016[Crossref], [PubMed], [CAS], Google Scholar275https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtVyitrnK&md5=87124626a1eceb76440c42fd87cc3360Use of fluorinated functionality in enzyme inhibitor development: Mechanistic and analytical advantagesBerkowitz, David B.; Karukurichi, Kannan R.; de la Salud-Bea, Roberto; Nelson, David L.; McCune, Christopher D.Journal of Fluorine Chemistry (2008), 129 (9), 731-742CODEN: JFLCAR; ISSN:0022-1139. (Elsevier B.V.)A review. On the one hand, owing to its electronegativity, relatively small size, and notable leaving group ability from anionic intermediates, fluorine offers unique opportunities for mechanism-based enzyme inhibitor design. On the other, the "bio-orthogonal" and NMR-active 19-fluorine nucleus allows the bioorg. chemist to follow the mechanistic fate of fluorinated substrate analogs or inhibitors as they are enzymically processed. This article takes an overview of the field, highlighting key developments along these lines. It begins by highlighting new screening methodologies for drug discovery that involve appropriate tagging of either the substrate or an array of potential substrates (i.e., in proteomics screens) with 19F-markers that then report back on turnover and function, resp., via the NMR screen. Taking this one step further, substrate-tagging with fluorine can be done in such a manner as to provide stereochem. information on enzyme mechanism. For example, substitution of one of the terminal hydrogens in phosphoenolpyruvate, provides insight into the, otherwise latent, facial selectivity of C-C bond formation in KDO synthase. Perhaps, most importantly, from the point of view of this discussion, appropriately tailored fluorinated functionality can be used to form stabilized "transition state analog" complexes with target enzymes. Thus, 5-fluorinated pyrimidines, α-fluorinated ketones, and 2-fluoro-2-deoxysugars each lead to covalent adduction of catalytic active site residues in thymidylate synthase (TS), serine protease and glycosidase enzymes, resp. In all such cases, 19F NMR allows the bioorg. chemist to spectrally follow "transition state analog" formation. Finally, the use of specific fluorinated functionality to engineer "suicide substrates" is highlighted in a discussion of the development of the α-(2'Z-fluoro)vinyl trigger for amino acid decarboxylase inactivation. Here 19F NMR allows the bioorg. chemist to glean useful partition ratio data directly from the NMR tube.276Conti, P.; Tamborini, L.; Pinto, A.; Blondel, A.; Minoprio, P.; Mozzarelli, A.; De Micheli, C. Drug discovery targeting amino acid racemases. Chem. Rev. 2011, 111, 6919– 6946, DOI: 10.1021/cr2000702[ACS Full Text], [CAS], Google Scholar276https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFGqurjL&md5=33df0c7a9ed05e54141c7b3f13203954Drug Discovery Targeting Amino Acid RacemasesConti, Paola; Tamborini, Lucia; Pinto, Andrea; Blondel, Arnaud; Minoprio, Paola; Mozzarelli, Andrea; De Micheli, CarloChemical Reviews (Washington, DC, United States) (2011), 111 (11), 6919-6946CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. In the following chapters we will discuss the classification of amino acid racemases, involved in the prodn. of D-amino acids through a racemization process of the L counterparts, their diffusion, function, localization, structural features and the state of the art in the discovery of novel drugs targeting such enzymes for the treatment of different pathologies.277Azam, M. A.; Jayaram, U. Inhibitors of alanine racemase enzyme: a review. J. Enzyme Inhib. Med. Chem. 2016, 31, 517– 526, DOI: 10.3109/14756366.2015.1050010[Crossref], [PubMed], [CAS], Google Scholar277https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmvFCmurg%253D&md5=26a4555f21c235bab0797409aec6902eInhibitors of alanine racemase enzyme: a reviewAzam, Mohammed Afzal; Jayaram, UnniJournal of Enzyme Inhibition and Medicinal Chemistry (2016), 31 (4), 517-526CODEN: JEIMAZ; ISSN:1475-6366. (Taylor & Francis Ltd.)Alanine racemase is a fold type III PLP-dependent amino acid racemase enzyme catalyzing the conversion of L-alanine to D-alanine utilized by bacterial cell wall for peptidoglycan synthesis. As there are no known homologs in humans, it is considered as an excellent antibacterial drug target. The std. inhibitors of this enzyme include O-carbamyl-D-serine, D-cycloserine, chlorovinyl glycine, alaphosphin, etc. D-Cycloserine is indicated for pulmonary and extra pulmonary tuberculosis but therapeutic use of drug is limited due to its severe toxic effects. Toxic effects due to off-target affinities of cycloserine and other substrate analogs have prompted new research efforts to identify alanine racemase inhibitors that are not substrate analogs. In this review, an updated status of known inhibitors of alanine racemase enzyme has been provided which will serve as a rich source of structural information and will be helpful in generating selective and potent inhibitor of alanine racemase.278Kollonitsch, J.; Barash, L.; Kahan, F. M.; Kropp, H. New antibacterial agent via photofluorination of a bacterial cell wall constituent. Nature 1973, 243, 346– 347, DOI: 10.1038/243346a0[Crossref], [PubMed], [CAS], Google Scholar278https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3sXkslCitb8%253D&md5=64452355855203c632f72c38e70ba7c8New antibacterial agent via photofluorination of a bacterial cell wall constituentKollonitsch, J.; Barash, L.; Kahan, F. M.; Kropp, H.Nature (London, United Kingdom) (1973), 243 (5406), 346-7CODEN: NATUAS; ISSN:0028-0836.A highly active antibacterial agent, which is active against gram-neg. and pos. bacteria, was prepd. by C-fluorination of a key component of the bacterial cell wall, D-alanine [338-69-2]. Thus, 3-fluoro-D-alanine [35455-20-0] at concns. of 6-100 μg/ml inhibited the growth of Escherichia coli. D-alanine at 6-100 μg/ml reversed the inhibitory effect of 3-fluoro-D-alanine at 25 μg/ml. The ED50 values of 3-fluoro-D-alanine for Streptococcus pyrogenes and Diplococcus pneumoniae in mice were 1.1 and 5 mg/kg, resp. Mice survived a single, oral dose of 2 g/kg.279Kollonitsch, J.; Barash, L. Organofluorine synthesis via photofluorination: 3-fluoro-d-alanine and 2-deuterio analogue, antibacterials related to the bacterial cell wall. J. Am. Chem. Soc. 1976, 98, 5591– 5593, DOI: 10.1021/ja00434a030[ACS Full Text], [CAS], Google Scholar279https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE28XlsVentro%253D&md5=15d16fed9c9c23e8206dbff5abf0cc71Organofluorine synthesis via photofluorination: 3-fluoro-D-alanine and 2-deuterio analog, antibacterials related to the bacterial cell wallKollonitsch, J.; Barash, L.Journal of the American Chemical Society (1976), 98 (18), 5591-3CODEN: JACSAT; ISSN:0002-7863.The antibacterials 3-fluoro-D-alanine (I) and its 2-deuterated version (II) were prepd. The design of I exploits a fundamental divergence in biosynthesis of the peptidoglycan component of the bacterial cell wall and of the metabolic pathways in humans. This divergence suggested application of the concept of antimetabolite synthesis via the specific approach of photofluorination. Thus, photofluorination of(D-alanine generated I which displays a high degree of antibacterial activity. A variant of I increased metabolic stability-and with unimpaired antibacterial activity-was obtained via photofluorination of 2-deuterio-D-alanine, namely 3-fluoro-D-alanine-2d (II), effective in vitro and in vivo against every bacterial strain tested.280Silverman, R. B.; Abeles, R. H. Inactivation of pyridoxal phosphate dependent enzymes by mono- and polyhaloalanines. Biochemistry 1976, 15, 4718– 4723, DOI: 10.1021/bi00666a028[ACS Full Text], [CAS], Google Scholar280https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2sXhsFyj&md5=4d136781d42b7741be7a8ebbf04de1e2Inactivation of pyridoxal phosphate dependent enzymes by mono- and polyhaloalaninesSilverman, Richard B.; Abeles, Robert H.Biochemistry (1976), 15 (21), 4718-23CODEN: BICHAW; ISSN:0006-2960.β,β-Dichloro- and β,β,β-trifluoroalanine irreversibly inactivate a no. of pyridoxal phosphate-dependent enzymes which catalyze β- or γ-elimination reactions. The inactivation is time dependent and the rate of inactivation is 1st order in enzyme concn. This suggests that inactivation is due to covalent modification of the enzyme by a species generated at the active site from the polyhaloalanine (i.e., suicide inactivation). Monohaloalanines are substrates and do not inactivate. For γ-cystathionase, covalent and stoichiometric attachment of β,β,β-trifluoroalanine-1-14C was shown. It is proposed that the mechanism of inactivation involves Schiff base formation between inactivator and enzyme-bound pyridoxal and subsequent elimination of HCl from dichloroalanine or HF from trifluoroalanine. This results in the formation of a β-halo-α,β unsatd. imine, an activated Michael acceptor. Michael addition of a nucleophile at the active site leads to covalent labeling of the enzyme and inactivation. Alanine racemase is also inactivated by the 2 polyhaloalanines. Glutamate-pyruvate and glutamate-oxalocetate transaminases are inactivated by monohaloalanines but not by polyhaloalanines.281Wang, E.; Walsh, C. Suicide substrates for the alanine racemase of Escherichia coli B. Biochemistry 1978, 17, 1313– 1321, DOI: 10.1021/bi00600a028[ACS Full Text], [CAS], Google Scholar281https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXktVahs7c%253D&md5=0d32c8dfec57779e35308e0c360e3f06Suicide substrates for the alanine racemase of Escherichia coli BWang, Elizabeth; Walsh, ChristopherBiochemistry (1978), 17 (7), 1313-21CODEN: BICHAW; ISSN:0006-2960.Alanine racemase was purified ∼9000-fold from E. coli and found to be a dimer of 100,000 daltons, contg. 1 mol. pyridoxal phosphate/subunit. The mol. basis for irrversible active site-directed inactivation of the enzyme by a no. of suicide substrates was examd. Both D and L isomers of β-fluoroalanine and β-chloroalanine partition between (a) α,β elimination to pyruvate, ammonia, and halide ion or (b) inactivation. No racemization is detectable. The Vmax for pyruvate formation from L-chloroalanine is ∼50-fold lower than from the D isomer of chloroalanine or either fluoroalanine. However, both enantiomeric pairs partition identically, ∼830 turnovers/inactivating event. This invariant partition ratio suggests that a common intermediate, the eneamino acid-pyridoxal phosphate complex, is the species responsible for inactivation, probably by Michael attack from a nucleophilic residue at the enzyme active site. In keeping with this idea, O-carbamoyl-D-serine and O-acetyl-D-serine also undergo enzyme-catalyzed elimination for 830 turnovers before causing irreversible inactivation, presumably from the same intermediate. In contrast, the L isomers of O-carbamoyl- or O-acetlyserine do not eliminate nor do they induce inactivation, but serve merely as reversible, competitive inhibitors of the enzyme. This suggests asym. binding regions for bulky β substituents at the active site and suggests D isomers of substituted β-alanines would be preferentially effective enzyme inactivators. D-Cycloserine also inactivates alanine racemase in time-dependent fashion. Thus, both natural antibiotics, O-carbamoyl-D-serine and D-cycloserine, previously reported as reversible alanine racemase inhibitors, are in fact suicide substrates along with the β-haloalanines.282Badet, B.; Roise, D.; Walsh, C. T. Inactivation of the dadB Salmonella typhimurium alanine racemase by D and L isomers of β-substituted alanines: kinetics, stoichiometry, active site peptide sequencing, and reaction mechanism. Biochemistry 1984, 23, 5188– 5194, DOI: 10.1021/bi00317a016[ACS Full Text], [CAS], Google Scholar282https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXlvVKru74%253D&md5=d55fa0aee74737d76947707917b98c66Inactivation of the dadB Salmonella typhimurium alanine racemase by D and L isomers of β-substituted alanines: kinetics, stoichiometry, active site peptide sequencing, and reaction mechanismBadet, Bernard; Roise, David; Walsh, Christopher T.Biochemistry (1984), 23 (22), 5188-94CODEN: BICHAW; ISSN:0006-2960.The pyridoxal phosphate-dependent S. typhimurium gene dadb alanine racemase (I) was inactivated with D- and L-β-fluoroalanine, D- and L-β-chloroalanine, and O-acetyl-D-serine. I inactivation with each isomer of β-chloro[14C]alanine followed by NaBH4 redn. and trypsin digestion afforded a single radiolabeled peptide. In the same manner, NaB3H4-reduced native I gave a single labeled peptide after trypsin digestion. Purifn. and sequencing of these 3 radioactive peptides revealed them to be a common, unique hexadecapeptide which contained labeled lysine at position 6 in each case. I which had been inactivated, but not reductively stabilized with NaBH4, released a labile pyridoxal phosphate-inactivator adduct on denaturation. The structure of this adduct suggested that I was inactivated by trapping the coenzyme in a ternary adduct with inactivator and the active-site lysine. Under denaturing conditions, facile α,β-elimination occurred, releasing the aldol adduct of pyruvate and pyridoxal phosphate. Redn. of the ternary enzyme adduct blocked this elimination pathway. The overall mechanism of I inactivation was discussed in light of these results.283Wang, E. A.; Walsh, C. Characteristics of β,β-difluoroalanine and β,β,β-trifluoroalanine as suicide substrates for Escherichia coli B alanine racemase. Biochemistry 1981, 20, 7539– 7546, DOI: 10.1021/bi00529a032[ACS Full Text], [CAS], Google Scholar283https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XivVSg&md5=d4db063b01c8f5861db1032c0ede2c5dCharacteristics of β,β-difluoroalanine and β,β,β-trifluoroalanine as suicide substrates for E. coli B alanine racemaseWang, Elizabeth A.; Walsh, ChristopherBiochemistry (1981), 20 (26), 7539-46CODEN: BICHAW; ISSN:0006-2960.Alanine racemase from Escherichia coli B has been shown to process D- and L-isomers of β-fluoroalanine as suicide substrates with an identical partitioning ratio for each enantiomer of 820 catalytic eliminations of HF per enzymic inactivation event, suggesting the aminoacrylate-pyridoxal phosphate (PLP) complex as a common, sym. partitioning species. In an attempt to systematically vary the partition ratio, an index of killing efficiency, the β,β-difluoroalanine and β,β,β-trifluoroalanine isomers were evaluated for substrate processing, suicidal inactivation kinetics, and partitioning ratio, and stability of inactive , derivatized enzyme forms. Both difluoroalanine isomers showed high Km values (116 mM for D, 102 mM for L) in catalytic HF loss to form fluoropyruvate. The Vmax for the D-isomer was ∼14-fold higher than that of the L-isomer. Limiting inactivation rate consts., calcd. from kcat and obsd. partition ratios of 5000 and 2600, resp., were 2.2 min-1 for D-difluoroalanine and 0.33 min-1 for L-difluoroalanine. For comparison, DL-trifluoroalanine turned over <10 times/enzyme mol. inactivated and was thus a very efficient suicide substrate. The est. inactivation rate const. was <1.0 min-1. These data were analyzed in terms of the partitioning behavior of the monofluoro- and difluoroaminoacrylate-PLP complexes as partitioning intermediates for turnover or for racemase inactivation. Whereas the mono- and trifluoroalanines yield stable inactive species the difluoroalanine isomers produced labile enzyme derivs., and regain of catalytic activity was analyzed in terms of the anticipated oxidn. state at the β-C atom of the substrate fragment adducted to the enzyme.284Faraci, W. S.; Walsh, C. T. Mechanism of inactivation of alanine racemase by β,β,β-trifluoroalanine. Biochemistry 1989, 28, 431– 437, DOI: 10.1021/bi00428a004[ACS Full Text], [CAS], Google Scholar284https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXltlGgtQ%253D%253D&md5=36b00328b07b93dbb9daff89ee5e8d3fMechanism of inactivation of alanine racemase by β,β,β-trifluoroalanineFaraci, W. Stephen; Walsh, Christopher T.Biochemistry (1989), 28 (2), 431-7CODEN: BICHAW; ISSN:0006-2960.The alanine racemases are a group of pyridoxal phosphate (PLP)-dependent bacterial enzymes that catalyze the racemization of alanine, providing D-alanine for cell wall synthesis. The inactivation of the alanine racemases from the Gram-neg. organism, Salmonella typhimurium, and the Gram-pos. organism, Bacillus stearothermophilus, with β,β,β-trifluoroalanine was studied. The inactivation occurred with the same rate const. as that for formation of a broad 460-490-nm chromophore. The loss of 2 F-/mol of inactivated enzyme and the retention of the [1-14C]trifluoroalanine label accompanied inhibition, suggesting a monofluoro enzyme adduct. Partial denaturation (1M guanidine) led to rapid return of the initial 420-nm chromophore, followed by a slower (t1/2 ∼30 min-1 h) loss of F- and 14CO2 release. At this point, redn. by NaB3H4 and tryptic digestion yielded a single radiolabeled peptide. The purifn. and sequencing of the peptide revealed that lysine-38 was covalently attached to the PLP cofactor. A mechanism for enzyme inactivation by trifluoroalanine is proposed and contrasted with earlier results on monohaloalanines, in which nucleophilic attack of released aminoacrylate on the PLP aldimine leads to enzyme inactivation. For trifluoroalanine inactivation, nucleophilic attack of lysine-38 on the electrophilic β-difluoro-α,β-unsatd. imine provides an alternative mode of inhibition for these enzymes.285Thornberry, N. A.; Bull, H. G.; Taub, D.; Wilson, K. E.; Giménez-Gallego, G.; Rosegay, A.; Soderman, D. D.; Patchett, A. A. Mechanism-based inactivation of alanine racemase by 3-halovinylglycines. J. Biol. Chem. 1991, 266, 21657– 21665[PubMed], [CAS], Google Scholar285https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXmslChu7Y%253D&md5=c9a822e5b5c446018a29c0d961411daaMechanism-based inactivation of alanine racemase by 3-halovinylglycinesThornberry, Nancy A.; Bull, Herbert G.; Taub, David; Wilson, Kenneth E.; Gimenez-Gallego, Guillermo; Rosegay, Avery; Soderman, Denis D.; Patchett, Arthur A.Journal of Biological Chemistry (1991), 266 (32), 21657-65CODEN: JBCHA3; ISSN:0021-9258.Alanine racemase, an enzyme important to bacterial cell wall synthesis, is irreversibly inactivated by 3-chloro- and 3-fluorovinylglycine. Using alanine racemase purified to homogeneity from Escherichia coli B, the efficient inactivation produced a lethal event for every 2.2 nonlethal turnovers, compared to 1 in 800 for fluoroalanine. The mechanism of inhibition involves enzyme-catalyzed halide elimination to form an allenic intermediate that partitions between reversible and irreversible covalent adducts, in the ratio 3:7. The reversible adduct (λmax = 516 nm) decays to regenerate free enzyme with a half-life of 23 min. The lethal event involves irreversible alkylation of a tyrosine residue in the sequence -Val-Gly-Tyr-Gly-Gly-Arg-: the second-order rate const. for this process with D-chlorovinylglycine (122 M-1 s-1), the most reactive analog examd., is faster than the equiv. rate const. for D-fluoroalanine (93 M-1 s-1). The high killing efficiency and fast turnover of these mechanism-based inhibitors suggest that their design, employing the haloethylene moiety to generate a reactive allene during catalysis, could be extended to provide useful inhibitors of a variety of enzymes that conduct carbanion chem.286Thornberry, N. A.; Bull, H. G.; Taub, D.; Greenlee, W. J.; Patchett, A. A.; Cordes, E. H. 3-Halovinylglycines. Efficient irreversible inhibitors of E. coli alanine racemase. J. Am. Chem. Soc. 1987, 109, 7543– 7544, DOI: 10.1021/ja00258a056[ACS Full Text], [CAS], Google Scholar286https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXisFGj&md5=e19af51c169636042c9fe6af3ee22d313-Halovinylglycines. Efficient irreversible inhibitors of Escherichia coli alanine racemaseThornberry, N. A.; Bull, H. G.; Taub, D.; Greenlee, W. J.; Patchett, A. A.; Cordes, E. H.Journal of the American Chemical Society (1987), 109 (24), 7543-4CODEN: JACSAT; ISSN:0002-7863.3-Halovinylglycines are a new class of potent mechanism-based irreversible inactivators of alanine racemase. Results with E. coli β-alanine racemase (I) indicate that nearly every catalytic event results in irreversible inactivation of the enzyme. In contrast, mechanism-based inactivation by previously studied β-substituted alanines is much less efficient, requiring ∼800 turnovers with this enzyme to produce 1 irreversible event. The incubation of homogeneous E. coli I with D-chlorovinylglycine, L-chlorovinylglycine, or DL-fluorovinylglycine resulted in irreversible inactivation of the enzyme as demonstrated by the inability of the enzyme to regain catalytic activity after prolonged dialysis. The inactivation kinetics were characterized by rapid, pseudo-1st-order irreversible inhibition of 70% of the enzyme. The 2nd-order rate const. that describes this initial inactivation for D-chlorovinylglycine (122 M-1 s-1) was comparable to the corresponding rate const. for 3-fluoro-D-alanine (93 M-1 s-1). The irreversible inhibition of the remaining enzyme was described by a 1st-order rate const. that was independent of inhibitor concn., halogen, and stereochem. (1.2 × 10-4 s-1). The fluorovinylglycines were 100-fold less reactive than the chlorovinylglycines. Consistent with the greater efficiency and more complex kinetics, there was phys. evidence that the halovinylglycines follow a different mechanism of inhibition than established for their simpler homologs, the β-substituted alanines.287Jung, M. J. Substrates and inhibitors of aromatic amino acid decarboxylase. Bioorg. Chem. 1986, 14, 429– 443, DOI: 10.1016/0045-2068(86)90007-6[Crossref], [CAS], Google Scholar287https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXmtlelsg%253D%253D&md5=d80ae494b692f22f889e5588bc98781fSubstrates and inhibitors of aromatic amino acid decarboxylaseJung, M. J.Bioorganic Chemistry (1986), 14 (4), 429-43CODEN: BOCMBM; ISSN:0045-2068.A review with 71 refs. on the reaction mechanism and specificity of the title mammalian decarboxylase with substrates, substrate analogs, and inhibitors.288Li, T.; Huo, L.; Pulley, C.; Liu, A. Decarboxylation mechanisms in biological system. Bioorg. Chem. 2012, 43, 2– 14, DOI: 10.1016/j.bioorg.2012.03.001[Crossref], [PubMed], [CAS], Google Scholar288https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtFajurvL&md5=ac3fa2546f673f01736184b01f6feb16Decarboxylation mechanisms in biological systemLi, Tingfeng; Huo, Lu; Pulley, Christopher; Liu, AiminBioorganic Chemistry (2012), 43 (), 2-14CODEN: BOCMBM; ISSN:0045-2068. (Elsevier B.V.)A review, examg. the mechanisms propelling cofactor-independent, org. cofactor-dependent and metal-dependent decarboxylase chem. Decarboxylation, the removal of carbon dioxide from org. acids, is a fundamentally important reaction in biol. Numerous decarboxylase enzymes serve as key components of aerobic and anaerobic carbohydrate metab. and amino acid conversion. In the past decade, knowledge of the mechanisms enabling these crucial decarboxylase reactions has continued to expand and inspire. This review focuses on the org. cofactors biotin, flavin, NAD, pyridoxal 5'-phosphate, pyruvoyl, and thiamin pyrophosphate as catalytic centers. Significant attention is also placed on the metal-dependent decarboxylase mechanisms.289Flynn, G. A.; Beight, D. W.; Bohme, E. H. W.; Metcalf, B. W. The synthesis of fluorinated aminophosphonic acid inhibitors of alanine racemase. Tetrahedron Lett. 1985, 26, 285– 288, DOI: 10.1016/S0040-4039(01)80798-X[Crossref], [CAS], Google Scholar289https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXltVGhtLY%253D&md5=20f34c6b8a9f12295b0e45cf9cde8021The synthesis of fluorinated aminophosphonic acid inhibitors of alanine racemaseFlynn, Gary A.; Beight, Douglas W.; Bohme, Ekkehard H. W.; Metcalf, Brian W.Tetrahedron Letters (1985), 26 (3), 285-8CODEN: TELEAY; ISSN:0040-4039.The synthesis of H2NCHRPO3H2 (I; R = CF3, CHF2, CH2F) from fluorinated acetic acids is described. I are potential inhibitors of alanine racemase which might exhibit the enhanced binding affinity and specificity of H2NCHMeCO2H while retaining the irreversible vector of the fluorinated alanines.290Kollonitsch, J.; Perkins, L. M.; Patchett, A. A.; Doldouras, G. A.; Marburg, S.; Duggan, D. E.; Maycock, A. L.; Aster, S. D. Selective inhibitors of biosynthesis of aminergic neurotransmitters. Nature 1978, 274, 906– 908, DOI: 10.1038/274906a0[Crossref], [PubMed], [CAS], Google Scholar290https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1MXhsVKhtrs%253D&md5=85391a6eca50f7a14b59c0e9084573caSelective inhibitors of biosynthesis of aminergic neurotransmittersKollonitsch, J.; Patchett, A. A.; Marburg, S.; Maycock, A. L.; Perkins, L. M.; Doldouras, G. A.; Duggan, D. E.; Aster, S. D.Nature (London, United Kingdom) (1978), 274 (5674), 906-8CODEN: NATUAS; ISSN:0028-0836.α-Fluoromethylamino acids and α-fluoromethyl amines were irreversible inhibitors of the amino acid decarboxylases involved in the formation of aminergic neurotransmitters. The inhibition of dopa decarboxylase (EC 4.1.1.26) [9042-64-2] by (S)-α-fluoromethyldopa [69672-34-0] was retarded by α-methyldopa, suggesting that the active site was the site of inactivation. Inhibition seemed to involve the stoichiometric formation of an enzyme-inhibitor adduct lacking the CO2H and F- moiety of the inhibitor.291Maycock, A. L.; Aster, S. D.; Patchett, A. A. Inactivation of 3-(3,4-dihydroxyphenyl)alanine decarboxylase by 2-(fluoromethyl)-3-(3,4-dihydroxyphenyl)alanine. Biochemistry 1980, 19, 709– 718, DOI: 10.1021/bi00545a016[ACS Full Text], [CAS], Google Scholar291https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3cXhtVKgtLo%253D&md5=8740361411508e1e694cd03ef873685cInactivation of 3-(3,4-dihydroxyphenyl)alanine decarboxylase by 2-(fluoromethyl)-3-(3,4-dihydroxyphenyl)alanineMaycock, A. L.; Aster, S. D.; Patchett, A. A.Biochemistry (1980), 19 (4), 709-18CODEN: BICHAW; ISSN:0006-2960.2-(Fluoromethyl)-3-(3,4-dihydroxyphenyl)alanine (I) caused rapid, time-dependent, stereospecific, and irreversible inhibition of hog kidney arom. amino acid (DOPA) decarboxylase. The inactivation occurred with loss of both the carboxyl C and F- from I and resulted in the stoichiometric formation of a covalent enzyme-inhibitor adduct. The data are consistent with I being a suicide inactivator of the enzyme, and a plausible mechanism for the inactivation process is presented. The inactivation is highly efficient in that there is essentially no enzymic turnover of I to produce the corresponding amine, 1-(fluoromethyl)-2-(3,4-dihydroxyphenyl)ethylamine (II). II was also a potent inactivator of the enzyme. In vivo, I inactivated both brain and peripheral (liver) DOPA decarboxylase activity. The possible significance of these data with respect to the known antihypertensive effect of I is discussed.292Metcalf, B. W.; Bey, P.; Danzin, C.; Jung, M. J.; Casara, P.; Vevert, J. P. Catalytic irreversible inhibition of mammalian ornithine decarboxylase (E.C. 4.1.1.17) by substrate and product analogues. J. Am. Chem. Soc. 1978, 100, 2551– 2553, DOI: 10.1021/ja00476a050[ACS Full Text], [CAS], Google Scholar292https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXktF2jsbg%253D&md5=eb551e94129f2c9ccf9c0821756fd060Catalytic irreversible inhibition of mammalian ornithine decarboxylase (E.C.4.1.1.17) by substrate and product analogsMetcalf, B. W.; Bey, P.; Danzin, C.; Jung, M. J.; Casara, P.; Vevert, J. P.Journal of the American Chemical Society (1978), 100 (8), 2551-3CODEN: JACSAT; ISSN:0002-7863.Ornithine analogs incorporating an α-Me group functionalized by fluoro, chloro, and cyano substituents, as well as the putrescine analogs 5-hexyne-1,4-diamine (I) and trans-hex-2-ene-5-yne-1,4-diamine (II), were catalytic irreversible inhibitors of a prepn. of mammaliam ornithine decarboxylase. For the ornithine analogs, it is proposed that decarboxylation of the enzyme-bound Schiff's base formed between pyridoxal phosphate and the analog leads to a reactive imine which can alkylate a nucleophilic residue at the enzyme's active site. Enzyme inactivation by I and II is rationalized on the basis of the microscopic reversibility principle. It is proposed that the proton abstraction implicit in the reverse reaction leads to a reactive allene in the active site, which, once again, is an alkylating agent.293Qu, N.; Ignatenko, N. A.; Yamauchi, P.; Stringer, D. E.; Levenson, C.; Shannon, P.; Perrin, S.; Gerner, E. W. Inhibition of human ornithine decarboxylase activity by enantiomers of difluoromethylornithine. Biochem. J. 2003, 375, 465– 470, DOI: 10.1042/bj20030382[Crossref], [PubMed], [CAS], Google Scholar293https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXnvF2rsLo%253D&md5=21fae1a86ab5b589ae285f47b0278b6dInhibition of human ornithine decarboxylase activity by enantiomers of difluoromethylornithineQu, Ning; Ignatenko, Natalia A.; Yamauchi, Phillip; Stringer, David E.; Levenson, Corey; Shannon, Patrick; Perrin, Scott; Gerner, Eugene W.Biochemical Journal (2003), 375 (2), 465-470CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)Racemic difluoromethylornithine (D/L-DFMO) is an inhibitor of ODC (ornithine decarboxylase), the first enzyme in eukaryotic polyamine biosynthesis. D/L-DFMO is an effective anti-parasitic agent and inhibitor of mammalian cell growth and development. Purified human ODC-catalyzed ornithine decarboxylation is highly stereospecific. However, both DFMO enantiomers suppressed ODC activity in a time- and concn.-dependent manner. ODC activity failed to recover after treatment with either L- or D-DFMO and dialysis to remove free inhibitor. The inhibitor dissocn. const. (K D) values for the formation of enzyme-inhibitor complexes were 28.3 ± 3.4, 1.3 ± 0.3 and 2.2 ± 0.4 μM resp. for D-, L- and D/L-DFMO. The differences in these K D values were statistically significant (P <0.05). The inhibitor inactivation consts. (K inact) for the irreversible step were 0.25 ± 0.03, 0.15 ± 0.03 and 0.15 ± 0.03 min-1 resp. for D-, L- and D/L-DFMO. These latter values were not statistically significantly different (P >0.1). D-DFMO was a more potent inhibitor (IC50 ∼7.5 μM) when compared with D-ornithine (IC50 ∼1.5 mM) of ODC-catalyzed L-ornithine decarboxylation. Treatment of human colon tumor-derived HCT116 cells with either L- or D-DFMO decreased the cellular polyamine contents in a concn.-dependent manner. These results show that both enantiomers of DFMO irreversibly inactivate ODC and suggest that this inactivation occurs by a common mechanism. Both enantiomers form enzyme-inhibitor complexes with ODC, but the probability of formation of these complexes is 20 times greater for L-DFMO when compared with D-DFMO. The rate of the irreversible reaction in ODC inactivation is similar for the L- and D-enantiomer. This unexpected similarity between DFMO enantiomers, in contrast with the high degree of stereospecificity of the substrate ornithine, appears to be due to the α-substituent of the inhibitor. The D-enantiomer may have advantages, such as decreased normal tissue toxicity, over L- or D/L-DFMO in some clin. applications.294Wallace, H. M.; Fraser, A. V.; Hughes, A. A perspective of polyamine metabolism. Biochem. J. 2003, 376, 1– 14, DOI: 10.1042/bj20031327[Crossref], [PubMed], [CAS], Google Scholar294https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXovVajs7Y%253D&md5=106d3c7e36e100d304d1f1a6570db3e0A perspective of polyamine metabolismWallace, Heather M.; Fraser, Alison V.; Hughes, AlunBiochemical Journal (2003), 376 (1), 1-14CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)A review. Polyamines are essential for the growth and function of normal cells. They interact with various macromols., both electrostatically and covalently and, as a consequence, have a variety of cellular effects. The complexity of polyamine metab. and the multitude of compensatory mechanisms that are invoked to maintain polyamine homeostasis argue that these amines are crit. to cell survival. The regulation of polyamine content within cells occurs at several levels, including transcription and translation. In addn., novel features such as the +1 frameshift required for antizyme prodn. and the rapid turnover of several of the enzymes involved in the pathway make the regulation of polyamine metab. a fascinating subject. The link between polyamine content and human disease is unequivocal, and significant success has been obtained in the treatment of a no. of parasitic infections. Targeting the polyamine pathway as a means of treating cancer has met with limited success, although the development of drugs such as DFMO (α-difluoromethylornithine), a rationally designed anticancer agent, has revolutionized our understanding of polyamine function in cell growth and provided proof of concept' that influencing polyamine metab. and content within tumor cells will prevent tumor growth. The more recent development of the polyamine analogs has been pivotal in advancing our understanding of the necessity to deplete all three polyamines to induce apoptosis in tumor cells. The current thinking is that the polyamine inhibitors/analogs may also be useful agents in the chemoprevention of cancer and, in this area, we may yet see a revival of DFMO. The future will be in adopting a functional genomics approach to identifying polyamine-regulated genes linked to either carcinogenesis or apoptosis.295Casero, R. A., Jr.; Murray Stewart, T.; Pegg, A. E. Polyamine metabolism and cancer: treatments, challenges and opportunities. Nat. Rev. Cancer 2018, 18, 681– 695, DOI: 10.1038/s41568-018-0050-3[Crossref], [PubMed], [CAS], Google Scholar295https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1Gks7nF&md5=9bcfc74df8e65e0ce8a53203770bd1f1Polyamine metabolism and cancer: treatments, challenges and opportunitiesCasero, Jr, Robert A.; Murray Stewart, Tracy; Pegg, Anthony E.Nature Reviews Cancer (2018), 18 (11), 681-695CODEN: NRCAC4; ISSN:1474-175X. (Nature Research)Advances in our understanding of the metab. and mol. functions of polyamines and their alterations in cancer have led to resurgence in the interest of targeting polyamine metab. as an anticancer strategy. Increasing knowledge of the interplay between polyamine metab. and other cancer-driving pathways, including the PTEN-PI3K-mTOR complex 1 (mTORC1), WNT signalling and RAS pathways, suggests potential combination therapies that will have considerable clin. promise. Addnl., an expanding no. of promising clin. trials with agents targeting polyamines for both therapy and prevention are ongoing. New insights into mol. mechanisms linking dysregulated polyamine catabolism and carcinogenesis suggest addnl. strategies that can be used for cancer prevention in at-risk individuals. In addn., polyamine blocking therapy, a strategy that combines the inhibition of polyamine biosynthesis with the simultaneous blockade of polyamine transport, can be more effective than therapies based on polyamine depletion alone and may involve an antitumor immune response. These findings open up new avenues of research into exploiting aberrant polyamine metab. for anticancer therapy.296Meyskens, F. L., Jr.; Gerner, E. W. Development of difluoromethylornithine as a chemoprevention agent for the management of colon cancer. J. Cell. Biochem. 1995, 59 (S22), 126– 131, DOI: 10.1002/jcb.240590816297Levin, V. A.; Ictech, S. E.; Hess, K. R. Clinical importance of eflornithine (α-difluoromethylornithine) for the treatment of malignant gliomas. CNS Oncol. 2018, 7, CNS16, DOI: 10.2217/cns-2017-0031298Ferrins, L.; Rahmani, R.; Baell, J. B. Drug discovery and human African trypanosomiasis: a disease less neglected?. Future Med. Chem. 2013, 5, 1801– 1841, DOI: 10.4155/fmc.13.162[Crossref], [PubMed], [CAS], Google Scholar298https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1CitLzK&md5=89e0f13e2bd1f3bdb4821ba513754069Drug discovery and human African trypanosomiasis: a disease less neglected?Ferrins, Lori; Rahmani, Raphael; Baell, Jonathan B.Future Medicinal Chemistry (2013), 5 (15), 1801-1841CODEN: FMCUA7; ISSN:1756-8919. (Future Science Ltd.)A review. Human African trypanosomiasis (HAT) has been neglected for a long time. The most recent drug to treat this disease, eflornithine, was approved by the US FDA in 2000. Current treatments exhibit numerous problematic side effects and are often ineffective against the debilitating CNS resident stage of the disease. Fortunately, several partnerships and initiatives have been formed over the last 20 years in an effort to eradicate HAT, along with a no. of other neglected diseases. This has led to an increasing no. of foundations and research institutions that are currently working on the development of new drugs for HAT and tools with which to diagnose and treat patients. New biochem. pathways as therapeutic targets are emerging, accompanied by increasing nos. of new antitrypanosomal compd. classes. The future looks promising that this collaborative approach will facilitate eagerly awaited breakthroughs in the treatment of HAT.299LoGiudice, N.; Le, L.; Abuan, I.; Leizorek, Y.; Roberts, S. C. α-Difluoromethylornithine, an irreversible inhibitor of polyamine biosynthesis, as a therapeutic strategy against hyperproliferative and infectious diseases. Med. Sci. 2018, 6, 12, DOI: 10.3390/medsci6010012[Crossref], [CAS], Google Scholar299https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtVKktrfO&md5=3c525e3ef0ec516b797fa8a7e66dec15Alpha-difluoromethylornithine, an irreversible inhibitor of polyamine biosynthesis, as a therapeutic strategy against hyperproliferative and infectious diseasesLoGiudice, Nicole; Le, Linh; Abuan, Irene; Leizorek, Yvette; Roberts, Sigrid C.Medical Sciences (2018), 6 (1), 12/1-12/17CODEN: MSECFJ; ISSN:2076-3271. (MDPI AG)The fluorinated ornithine analog -difluoromethylornithine (DFMO, eflornithine, ornidyl) is an irreversible suicide inhibitor of ornithine decarboxylase (ODC), the first and rate-limiting enzyme of polyamine biosynthesis. The ubiquitous and essential polyamines have many functions, but are primarily important for rapidly proliferating cells. Thus, ODC is potentially a drug target for any disease state where rapid growth is a key process leading to pathol. The compd. was originally discovered as an anticancer drug, but its effectiveness was disappointing. However, DFMO was successfully developed to treat African sleeping sickness and is currently one of few clin. used drugs to combat this neglected tropical disease. The other Food and Drug Administration (FDA) approved application for DFMO is as an active ingredient in the hair removal cream Vaniqa. In recent years, renewed interest in DFMO for hyperproliferative diseases has led to increased research and promising preclin. and clin. trials. This review explores the use of DFMO for the treatment of African sleeping sickness and hirsutism, as well as its potential as a chemopreventive and chemotherapeutic agent against colorectal cancer and neuroblastoma.300Coyne, P. E., Jr. The eflornithine story. J. Am. Acad. Dermatol. 2001, 45, 784– 786, DOI: 10.1067/mjd.2001.117853[Crossref], [PubMed], [CAS], Google Scholar300https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3MrmtVOhtw%253D%253D&md5=374124f20a8698771d87bc950b3d54f7The eflornithine storyCoyne P E JrJournal of the American Academy of Dermatology (2001), 45 (5), 784-6 ISSN:0190-9622.There is no expanded citation for this reference.301McCune, C. D.; Beio, M. L.; Sturdivant, J. M.; de la Salud-Bea, R.; Darnell, B. M.; Berkowitz, D. B. Synthesis and deployment of an elusive fluorovinyl cation equivalent: Access to quaternary α-(1′-fluoro)vinyl amino acids as potential PLP enzyme inactivators. J. Am. Chem. Soc. 2017, 139, 14077– 14089, DOI: 10.1021/jacs.7b04690[ACS Full Text], [CAS], Google Scholar301https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsV2jsrnE&md5=1322ac88e9c11007dfb9d820a7a1d875Synthesis and deployment of an elusive fluorovinyl cation equivalent: Access to quaternary α-(1'-fluoro)vinyl amino acids as potential PLP enzyme inactivatorsMcCune, Christopher D.; Beio, Matthew L.; Sturdivant, Jill M.; de la Salud-Bea, Roberto; Darnell, Brendan M.; Berkowitz, David B.Journal of the American Chemical Society (2017), 139 (40), 14077-14089CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Developing specific chem. functionalities to deploy in biol. environments for targeted enzyme inactivation lies at the heart of mechanism-based inhibitor development but also is central to other protein-tagging methods in modern chem. biol. including activity-based protein profiling and proteolysis-targeting chimeras. We describe here a previously unknown class of potential PLP enzyme inactivators; namely, a family of quaternary, α-(1'-fluoro)vinyl amino acids, bearing the side chains of the cognate amino acids. These are obtained by the capture of suitably protected amino acid enolates with β,β-difluorovinyl Ph sulfone, a new (1'-fluoro)vinyl cation equiv., and an electrophile that previously eluded synthesis, capture and characterization. A significant variety of biol. relevant AA side chains are tolerated including those for alanine, valine, leucine, methionine, lysine, phenylalanine, tyrosine, and tryptophan. Following addn./elimination, the resulting transoid α-(1'-fluoro)-β-(phenylsulfonyl)vinyl AA-esters undergo smooth sulfone-stannane interchange to stereoselectively give the corresponding transoid α-(1'-fluoro)-β-(tributylstannyl)vinyl AA-esters. Protodestannylation and global deprotection then yield these sterically encumbered and densely functionalized quaternary amino acids. The α-(1'-fluoro)vinyl trigger, a potential allene-generating functionality originally proposed by Abeles, is now available in a quaternary AA context for the first time. In an initial test of this new inhibitor class, α-(1'-fluoro)vinyllysine is seen to act as a time-dependent, irreversible inactivator of lysine decarboxylase from Hafnia alvei. The enantiomers of the inhibitor could be resolved, and each is seen to give time-dependent inactivation with this enzyme. Kitz-Wilson anal. reveals similar inactivation parameters for the two antipodes, L-α-(1'-fluoro)vinyllysine (Ki = 630 ± 20 μM; t1/2 = 2.8 min) and D-α-(1'-fluoro)vinyllysine (Ki = 470 ± 30 μM; t1/2 = 3.6 min). The stage is now set for exploration of the efficacy of this trigger in other PLP-enzyme active sites.302Abeles, R. H.; Alston, T. A. Enzyme inhibition by fluoro compounds. J. Biol. Chem. 1990, 265, 16705– 16708[PubMed], [CAS], Google Scholar302https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXisVKh&md5=9b91b5367f5c8085a9b00e90d0e81d37Enzyme inhibition by fluoro compoundsAbeles, Robert H.; Alston, Theodore A.Journal of Biological Chemistry (1990), 265 (28), 16705-8CODEN: JBCHA3; ISSN:0021-9258.A review, with 91 refs., on F in enzyme inhibitors (including drugs and toxins). F properties (i.e. steric compactness and electronegativity) are considered with regard to replacement of H and OH groups, inductive effects, and function without F as a leaving group.303Xu, Y.; Abeles, R. H. Inhibition of tryptophan synthase by (1-fluorovinyl)glycine. Biochemistry 1993, 32, 806– 811, DOI: 10.1021/bi00054a010[ACS Full Text], [CAS], Google Scholar303https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXlsV2hsg%253D%253D&md5=502800786642e33ed3f4d5fa1b97ef30Inhibition of tryptophan synthase by (1-fluorovinyl)glycineXu, Yajun; Abeles, Robert H.Biochemistry (1993), 32 (3), 806-11CODEN: BICHAW; ISSN:0006-2960.Tryptophan synthase (α2β2 complex) from Salmonella typhimurium catalyzes the formation of tryptophan from serine and indole. The enzyme is inactivated by (1-fluorovinyl)glycine. Concomitant with enzyme inactivation, the absorbance at 485 nm increases, indicating covalent modification of pyridoxal 5'-phosphate. It is proposed that inactivation involves elimination of HF to form an allene, which reacts with a nucleophile at the active site. The inactivation reaction involves an α,β-elimination, as does the formation of tryptophan from indole and serine. The inactivation occurs with kin > 1.3 s-1, which is very close to kcat (6.4 s-1) for the formation of tryptophan from indole and serine. The inactive enzyme (α2β2) regains activity with koff = 0.005 min-1. Aminoacetone is formed during reactivation, and pyridoxal 5'-phosphate is regenerated. Tryptophan synthase also catalyzes the dehydration of serine, or 3-fluoroalanine, to pyruvate in the absence of indole. This reaction involves an α,β-elimination and the intermediate formation of an aminoacrylate adduct with pyridoxal 5'-phosphate, as does the formation of tryptophan. Pyruvate formation proceeds at less than 5% the rate of tryptophan formation. With [2-2H]serine an isotope effect (DVmax = 1.5) is obsd. It is proposed that pyruvate formation is limited by the rate of hydration of the aminoacrylate intermediate and the rate of the abstraction of the serine α-hydrogen.304Silverman, R. B.; Levy, M. A. Substituted 4-aminobutanoic acids. Substrates for γ-aminobutyric acid α-ketoglutaric acid aminotransferase. J. Biol. Chem. 1981, 256, 11565– 11568[PubMed], [CAS], Google Scholar304https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXmtFKqtLw%253D&md5=c85d3103bf565c075232641eebacc8feSubstituted 4-aminobutanoic acids. Substrates for γ-aminobutyric acid α-ketoglutaric acid aminotransferaseSilverman, Richard B.; Levy, Mark A.Journal of Biological Chemistry (1981), 256 (22), 11565-8CODEN: JBCHA3; ISSN:0021-9258.Substituted 4-aminobutanoic acids were studied as potential irreversible inactivators of purified pig brain γ-aminobutyrate (GABA) transaminase, the enzyme responsible for the degrdn. of the inhibitory neurotransmitter, GABA. Unlike the related 4-amino-5-halopentanoic acids, the 4-amino-3-halobutanoic acids are substrates for this enzyme, undergoing exclusive elimination to succinic semialdehyde and producing no inactivation. The hydroxy analog, however, undergoes exclusive transamination and no succinic semialdehyde is detected. These results are discussed in terms of the nature of the substituents, the structure of the active site of GABA transaminase, and the design of mechanism-based inactivators.305Clift, M. D.; Ji, H.; Deniau, G. P.; O’Hagan, D.; Silverman, R. B. Enantiomers of 4-amino-3-fluorobutanoic acid as substrates for γ-aminobutyric acid aminotransferase. Conformational probes for GABA binding. Biochemistry 2007, 46, 13819– 13828, DOI: 10.1021/bi701249q[ACS Full Text], [CAS], Google Scholar305https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1OhsbrO&md5=40d7bf78aab98245d752399c4f468bf1Enantiomers of 4-Amino-3-fluorobutanoic Acid as Substrates for γ-Aminobutyric Acid Aminotransferase. Conformational Probes for GABA BindingClift, Michael D.; Ji, Haitao; Deniau, Gildas P.; O'Hagan, David; Silverman, Richard B.Biochemistry (2007), 46 (48), 13819-13828CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)γ-Aminobutyric acid aminotransferase (GABA-AT), a pyridoxal 5'-phosphate dependent enzyme, catalyzes the degrdn. of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) to succinic semialdehyde with concomitant conversion of pyridoxal 5'-phosphate (PLP) to pyridoxamine 5'-phosphate (PMP). The enzyme then catalyzes the conversion of α-ketoglutarate to the excitatory neurotransmitter L-glutamate. Racemic 4-amino-3-fluorobutanoic acid (3-F-GABA) was shown previously to act as a substrate for GABA-AT, not for transamination, but for HF elimination. Here we report studies of the reaction catalyzed by GABA-AT on (R)- and (S)-3-F-GABA. Neither enantiomer is a substrate for transamination. Very little elimination from the (S)-enantiomer was detected using a coupled enzyme assay; The rate of elimination of HF from the (R)-enantiomer is at least 10 times greater than that for the (S)-enantiomer. The (R)-enantiomer is about 20 times more efficient as a substrate for GABA-AT catalyzed HF elimination than GABA is a substrate for transamination. The (R)-enantiomer also inhibits the transamination of GABA 10 times more effectively than the (S)-enantiomer. Using a combination of computer modeling and the knowledge that vicinal C-F and C-NH3+ bonds have a strong preference to align gauche rather than anti to each other, it is concluded that on binding of free 3-F-GABA to GABA-AT the optimal conformation places the C-NH3+ and C-F bonds gauche in the (R)-enantiomer but anti in the (S)-enantiomer. Furthermore, the dynamic binding process and the bioactive conformation of GABA bound to GABA-AT have been inferred on the basis of the different biol. behavior of the two enantiomers of 3-F-GABA when they bind to the enzyme. The present study suggests that the C-F bond can be utilized as a conformational probe to explore the dynamic binding process and provide insight into the bioactive conformation of substrates, which cannot be easily detd. by other biophys. approaches.306Deniau, G.; Slawin, A. M.; Lebl, T.; Chorki, F.; Issberner, J. P.; van Mourik, T.; Heygate, J. M.; Lambert, J. J.; Etherington, L. A.; Sillar, K. T.; O’Hagan, D. Synthesis, conformation and biological evaluation of the enantiomers of 3-fluoro-γ-aminobutyric acid ((R)- and (S)-3F-GABA): an analogue of the neurotransmitter GABA. ChemBioChem 2007, 8, 2265– 2274, DOI: 10.1002/cbic.200700371[Crossref], [PubMed], [CAS], Google Scholar306https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXltlentbc%253D&md5=9ebd7a5dc80ad3dd9c711572f1304f32Synthesis, conformation and biological evaluation of the enantiomers of 3-fluoro-γ-aminobutyric acid ((R)- and (S)-3F-GABA): an analog of the neurotransmitter GABADeniau, Gildas; Slawin, Alexandra M. Z.; Lebl, Tomas; Chorki, Fatima; Issberner, Jon P.; van Mourik, Tanja; Heygate, Judith M.; Lambert, Jeremy J.; Etherington, Lori-An; Sillar, Keith T.; O'Hagan, DavidChemBioChem (2007), 8 (18), 2265-2274CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)β-Aminobutyric acid or GABA (1) is one of the major inhibitory amino acid neurotransmitters of the central nervous system. This article describes the first synthesis of both the (R)- and (S)- enantiomers of 3-fluoro-GABA (2, 3F-GABA). DFT calcns. were carried out in a continuum solvent model (PCM-B3LYP) to est. the preferred conformations of 3F-GABA in aq. soln. NMR coupling consts. were calcd. for each conformer and were then used to simulate the NMR spectra to evaluate the soln. conformation of 3F-GABA. A preliminary evaluation of the 3F-GABA enantiomers shows that they act similarly as agonists of cloned GABAA receptors; however, they behave quite differently in a whole animal (Xenopus laevis tadpole model).307Buissonneaud, D. Y.; van Mourik, T.; O’Hagan, D. A DFT study on the origin of the fluorine gauche effect in substituted fluoroethanes. Tetrahedron 2010, 66, 2196– 2202, DOI: 10.1016/j.tet.2010.01.049[Crossref], [CAS], Google Scholar307https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXitleiurk%253D&md5=b7d6e3599c8e0b9a6364f55c34f362afA DFT study on the origin of the fluorine gauche effect in substituted fluoroethanesBuissonneaud, David Y.; van Mourik, Tanja; O'Hagan, DavidTetrahedron (2010), 66 (12), 2196-2202CODEN: TETRAB; ISSN:0040-4020. (Elsevier Ltd.)DFT derived conformational energy profiles of a series of β-substituted α-fluoroethanes (F-CH2CH2-X) have been explored where the substituent X was varied as NH3+, OCOH, NCO, NO2, NHCHO, F, N3, CH=NH, NCS, CH=C=CH2, CH3, CH=CH2, NC, CN, CHO, and CCH. Comparisons were correlated relative to 1,2-difluoroethane, a compd. which exhibits a well known gauche preference. Only four of the compds. displayed an anti preference, with the large majority preferring a gauche conformation. In particular the influence of steric and electrostatic attraction/repulsion between the fluorine atom and the X-substituent was explored by evaluating rotational energy profiles for all compds. and sep. NBO correlations were evaluated to assess the contribution of hyperconjugation to the minimized gauche and anti conformers. In the event the gauche preference for 1,2-difluoroethane was shown to have an origin due largely to σ(C-H)→σ*(C-F) hyperconjugative interactions, whereas the conformational preference for the remaining structures is rationalized by hyperconjugative as well as steric and electrostatic contributions. The anti preferred compds. 13, 14 and 16 possessed triple bonds and the preference arose due to fluorine/p-orbital repulsion.308Boeckxstaens, G. E.; Denison, H.; Jensen, J. M.; Lehmann, A.; Ruth, M. Translational gastrointestinal pharmacology in the 21st century: ’the lesogaberan story. Curr. Opin. Pharmacol. 2011, 11, 630– 633, DOI: 10.1016/j.coph.2011.10.011[Crossref], [PubMed], [CAS], Google Scholar308https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFegsbbF&md5=8e8fb9878270ac3f1d68bd6ad2b2b4deTranslational gastrointestinal pharmacology in the 21st century: the lesogaberan story'Boeckxstaens, Guy E.; Denison, Hans; Jensen, Joergen M.; Lehmann, Anders; Ruth, MagnusCurrent Opinion in Pharmacology (2011), 11 (6), 630-633CODEN: COPUBK; ISSN:1471-4892. (Elsevier Ltd.)A review. The development of the novel γ-aminobutyric acid type-B receptor (GABAB) agonist lesogaberan is presented as an example of a partly successful translational strategy in the field of gastroenterol. Data on transient lower esophageal sphincter relaxations (TLESRs) and gastroesophageal reflux inhibition from preclin. models translated well to clin. studies in healthy volunteers and patients with gastroesophageal reflux disease (GERD). Animal models have also been used successfully to predict the effect of other target mechanisms on TLESRs in humans. However, while translation of physiol. to symptomatol. in patients with GERD was achieved, the effect size was too small to be of clin. significance. A deeper understanding of the cause of symptoms in different patient categories is therefore required.309Ekdahl, A.; Aurell-Holmberg, A.; Castagnoli, N., Jr. Identification of the metabolites of lesogaberan using linear trap quadrupole orbitrap mass spectrometry and hydrophilic interaction liquid chromatography. Xenobiotica 2013, 43, 461– 467, DOI: 10.3109/00498254.2012.725486[Crossref], [PubMed], [CAS], Google Scholar309https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXltVOqsrk%253D&md5=bf71cca5a686d1bee441e1ce842647caIdentification of the metabolites of lesogaberan using linear trap quadrupole orbitrap mass spectrometry and hydrophilic interaction liquid chromatographyEkdahl, Anja; Aurell-Holmberg, Ann; Castagnoli, Neal, Jr.Xenobiotica (2013), 43 (5), 461-467CODEN: XENOBH; ISSN:0049-8254. (Informa Healthcare)In this study, hydrophilic interaction liq. chromatog. (HILIC), radiochem. activity monitoring and linear trap quadrupole orbitrap mass spectrometry (MS) and tandem mass spectrometry (MS/MS) were used to identify the metabolites of a highly polar novel γ-aminobutyric acid type-B receptor agonist, lesogaberan, in rats. Urine was collected from 3 male Wistar rats for 24 h after dosing with 14C-labeled lesogaberan (170 mg/kg, 10 MBq/kg); blood plasma samples were taken 2 and 24 h after dosing. Pooled samples were sepd. by HILIC and eluents were analyzed by radiochem. activity monitoring, MS and MS/MS. Only the parent compd. was detected in plasma, but 6 metabolites (M1-M6) were detected in urine. Anal. of MS and MS/MS data and comparison with synthetic ref. stds. enabled the identification of the structure of each metabolite. M1 was identified as the N-acetylated species [(2R)-3-acetamido-2-fluoropropyl]-phosphinic acid, and M6 as [(2R)-3-amino-2-fluoropropyl]-phosphonic acid. Metabolites M2 and M5 were the alc. and carboxylic acid species 3-hydroxypropyl-phosphinic acid and 3-hydroxyphosphonoyl-propanoic acid, resp., both of which had lost the fluorine atom present in the parent compd. M3 was the corresponding carboxylic acid species retaining the fluorine atom, (2R)-2-fluoro-3-hydroxyphosphonoyl-propanoic acid. Finally M4 was identified as [(2R)-2-fluoro-3-guanidino-propyl]-phosphinic acid.310Lehmann, A.; Antonsson, M.; Holmberg, A. A.; Blackshaw, L. A.; Branden, L.; Brauner-Osborne, H.; Christiansen, B.; Dent, J.; Elebring, T.; Jacobson, B. M.; Jensen, J.; Mattsson, J. P.; Nilsson, K.; Oja, S. S.; Page, A. J.; Saransaari, P.; von Unge, S. R)-(3-amino-2-fluoropropyl) phosphinic acid (AZD3355), a novel GABAB receptor agonist, inhibits transient lower esophageal sphincter relaxation through a peripheral mode of action. J. Pharmacol. Exp. Ther. 2009, 331, 504– 512, DOI: 10.1124/jpet.109.153593[Crossref], [PubMed], [CAS], Google Scholar310https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlKnu7zI&md5=3f342dd2efa576ec5b3362ece793594f(R)-(3-amino-2-fluoropropyl) phosphinic acid (AZD3355), a novel GABAB receptor agonist, inhibits transient lower esophageal sphincter relaxation through a peripheral mode of actionLehmann, Anders; Antonsson, Madeleine; Holmberg, Ann Aurell; Blackshaw, L. Ashley; Braenden, Lena; Braeuner-Osborne, Hans; Christiansen, Bolette; Dent, John; Elebring, Thomas; Jacobson, Britt-Marie; Jensen, Joergen; Mattsson, Jan P.; Nilsson, Karolina; Oja, Simo S.; Page, Amanda J.; Saransaari, Pirjo; von Unge, SverkerJournal of Pharmacology and Experimental Therapeutics (2009), 331 (2), 504-512CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)Gastroesophageal reflux disease (GERD) affects >10% of the Western population. Conventionally, GERD is treated by reducing gastric acid secretion, which is effective in most patients but inadequate in a significant minority. We describe a new therapeutic approach for GERD, based on inhibition of transient lower esophageal sphincter relaxation (TLESR) with a proposed peripherally acting GABAB receptor agonist, (R)-(3-amino-2-fluoropropyl)phosphinic acid (AZD3355). AZD3355 potently stimulated recombinant human GABAB receptors and inhibited TLESR in dogs, with a biphasic dose-response curve. In mice, AZD3355 produced considerably less central side effects than the prototypical GABAB receptor agonist baclofen but evoked hypothermia at very high doses (blocked by a GABAB receptor antagonist and absent in GABAB-/- mice). AZD3355 and baclofen differed markedly in their distribution in rat brain; AZD3355, but not baclofen, was concd. in circumventricular organs as a result of active uptake (shown by avid intracellular sequestration) and related to binding of AZD3355 to native GABA transporters in rat cerebrocortical membranes. AZD3355 was also shown to be transported by all four recombinant human GABA transporters. AR-H061719 [(R/S)-(3-amino-2-fluoropropyl)phosphinic acid], (the racemate of AZD3355) inhibited the response of ferret mechanoreceptors to gastric distension, further supporting its peripheral site of action on TLESR. In summary, AZD3355 probably inhibits TLESR through stimulation of peripheral GABAB receptors and may offer a potential new approach to treatment of GERD.311Darland, G. K.; Hajdu, R.; Kropp, H.; Kahan, F. M.; Walker, R. W.; Vandenheuvel, W. J. Oxidative and defluorinative metabolism of fludalanine, 2-2H-3-fluoro-d-alanine. Drug Metab. Dispos. 1986, 14, 668– 673[PubMed], [CAS], Google Scholar311https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXnvVWltA%253D%253D&md5=554409be533e8ce74036e9ca2cf19dfbOxidative and defluorinative metabolism of fludalanine, 2-2H-3-fluoro-D-alanineDarland, Gary K.; Hajdu, Richard; Kropp, Helmut; Kahan, Frederick M.; Walker, Robert W.; Vandenheuvel, William J. A.Drug Metabolism and Disposition (1986), 14 (6), 668-73CODEN: DMDSAI; ISSN:0090-9556.The antibacterial agent fludalanine [35523-45-6] (used in this study as the deuterated analog 2-2H-3-fluoro-D-alanine (DFA) [35523-45-6]) is a potent inhibitor of bacterial alanine racemase, an enzyme required for the generation of D-alanine, an essential component of the bacterial cell wall. Primary metab. of DFA involves its oxidn. to fluoropyruvate (FP) [433-48-7]; this org. F- is then rapidly reduced to fluorolactate (FL) [433-47-6], which is the major org. metabolite in lab. animals. Gas-liq. chromatog. chem. ionization mass spectrometric assays were developed for these 2 metabolites. FL is the predominant organofluoride metabolite of DFA in the circulation. FP was detected in the urine although recovery was very low. The rapid conversion of FP to FL precludes assay of the former in serum. Max. serum FL concns. in the rat appear about 1 h after the dose of DFA and are relatively const. for several hours thereafter. The peak FL concn. is proportional to the dose of DFA; repeated daily dosing of DFA appears to cause neither satn. nor induction of metabolic pathways. Comparison of FL concns. detd. using the GC/MS assay with those based on an enzymic method specific for L-(+)-FL [3130-92-5] demonstrated that only the latter isomer is found in the plasma of monkeys dosed with DFA. In vivo exchange studies involving the α-proton of FL indicate that a small FP pool exists and is in equil. with FL. A crude pyruvate dehydrogenase [9014-20-4] complex isolated from beef heart mitochondria was shown to produce equimolar quantities of acetate [64-19-7], CO2, and F- from FP.312Patel, H.; Nemeria, N. S.; Andrews, F. H.; McLeish, M. J.; Jordan, F. Identification of charge transfer transitions related to thiamin-bound intermediates on enzymes provides a plethora of signatures useful in mechanistic studies. Biochemistry 2014, 53, 2145– 2152, DOI: 10.1021/bi4015743[ACS Full Text], [CAS], Google Scholar312https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXktlSrt70%253D&md5=4c355daabd03dde83b9ae902d7a39844Identification of charge transfer transitions related to thiamin-bound intermediates on enzymes provides a plethora of signatures useful in mechanistic studiesPatel, Hetalben; Nemeria, Natalia S.; Andrews, Forest H.; McLeish, Michael J.; Jordan, FrankBiochemistry (2014), 53 (13), 2145-2152CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The identification of enzyme-bound intermediates via their spectroscopic signatures, which then allows direct monitoring of the kinetic fate of these intermediates, poses a continuing challenge. As an electrophilic covalent catalyst, the thiamin diphosphate (ThDP) coenzyme forms a no. of noncovalent and covalent intermediates along its reaction pathways, and multiple UV-visible and CD spectroscopic bands have been identified by the authors pertinent to several among them. These electronic transitions fall into 2 classes: those for which the conjugated system provides a reasonable guide to the obsd. λmax and others in which there is no corresponding conjugated system and the obsd. CD bands are best ascribed to charge transfer (CT) transitions. Here, the authors report the reaction of 4 ThDP-dependent enzymes (pyruvate decarboxylase, pyruvate and oxoglutarate dehydrogenase complex E1 components, and benzaldehyde lyase) with alternate substrates: (a) acetylpyruvate, its Me ester, and fluoropyruvate, these providing the shortest side-chains attached at the thiazolium C2 atom and leading to CT bands with λmax values of >390 nm, not pertinent to any on-pathway conjugated systems (estd. λmax values of <330 nm), and (b) (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid displaying both a conjugated enamine (430 nm) and a CT transition (480 nm). The authors suggest that the CT transitions result from an interaction of the π bond on the ThDP C2 side-chain as a donor, and the pos. charged thiazolium ring as an acceptor, and correspond to covalent ThDP-bound intermediates. Time resoln. of these bands allows the rate consts. for individual steps to be detd. These CD methods can be applied to the entire ThDP superfamily of enzymes and should find applications with other enzymes.313Cordes, E. E. Chapter 11. Fludalanine. Nice try but no hallelujah. In Hallelujah Moments: Tales of Drug Discovery; Oxford University Press: New York, 2014; pp 183– 196.314Zhang, D.; Ogan, M.; Gedamke, R.; Roongta, V.; Dai, R.; Zhu, M.; Rinehart, J. K.; Klunk, L.; Mitroka, J. Protein covalent binding of Maxipost through a cytochrome P450-mediated ortho-quinone methide intermediate in rats. Drug Metab. Dispos. 2003, 31, 837– 845, DOI: 10.1124/dmd.31.7.837[Crossref], [PubMed], [CAS], Google Scholar314https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXltFalu7s%253D&md5=db2e7b4ad296af0226ac6d8b6b7b37adProtein covalent binding of maxipost through a cytochrome P450-mediated ortho-quinone methide intermediate in ratsZhang, Donglu; Ogan, Marc; Gedamke, Richard; Roongta, Vikram; Dai, Renke; Zhu, Mingshe; Rinehart, J. Kent; Klunk, Lewis; Mitroka, JamesDrug Metabolism and Disposition (2003), 31 (7), 837-845CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)(3S)-(+)-(5-Chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-((trifluoromethyl)-2H-indole-2-one) (MaxiPost, BMS-204352) is a potent and specific opener for maxi-K channels and has potential to prevent and treat ischemic stroke. Following single i.v. doses of [14C]BMS-204352 to rats, only 10 to 12% of radioactivity was extractable from plasma with org. solvents. The unextractable radioactivity remained assocd. with the proteins (mostly albumin) after SDS-polyacrylamide gel electrophoresis or dialysis. Following acid hydrolysis in 6 M HCl for 24 h at 110° from plasma proteins collected from nine rats dosed with [14C]BMS-204352, one major radioactive product was isolated and identified as a lysine-adduct of des-fluoro des-O-Me BMS-204352 by liq. chromatog./mass spectrometry and NMR analyses as well as by comparison with the synthetic analog, lysine-adduct of des-fluoro BMS-204352 (BMS-349821). The covalent binding of BMS-204352 results from the displacement of the ring-fluorine atom of des-O-Me BMS-204352 with the ε-amino group of a lysine residue. Microsomal incubations of [14C]BMS-204352 resulted in low levels of covalent binding of radioactivity to proteins. This in vitro covalent binding required cytochrome P 450-reductase cofactor NADPH and was attenuated by glutathione. P 4503A inhibitors ketoconazole and troleadomycin selectively prevented the covalent binding in vitro. Based on these observations, a two-step bioactivation process for the protein covalent binding of BMS-204352 was postulated:. (1) P 4503A-mediated O-demethylation leading to spontaneous release of HF and the formation of an ortho-quinone methide reactive metabolite and. (2) Nucleophilic addn. of the ε-amino group of protein lysine residue(s) in protein to form des-fluoro des-O-Me BMS-204352 lysine adduct.315Hewawasam, P.; Gribkoff, V. K.; Pendri, Y.; Dworetzky, S. I.; Meanwell, N. A.; Martinez, E.; Boissard, C. G.; Post-Munson, D. J.; Trojnacki, J. T.; Yeleswaram, K.; Pajor, L. M.; Knipe, J.; Gao, Q.; Perrone, R.; Starrett, J. E., Jr. The synthesis and characterization of BMS-204352 (MaxiPost) and related 3-fluorooxindoles as openers of maxi-K potassium channels. Bioorg. Med. Chem. Lett. 2002, 12, 1023– 1026, DOI: 10.1016/S0960-894X(02)00101-4[Crossref], [PubMed], [CAS], Google Scholar315https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XitFWhsLw%253D&md5=445a2b24bc60ad13a70a421d201a91d9The synthesis and characterization of BMS-204352 (MaxiPost) and related 3-fluorooxindoles as openers of maxi-K potassium channelsHewawasam, Piyasena; Gribkoff, Valentin K.; Pendri, Yadagiri; Dworetzky, Steven I.; Meanwell, Nicholas A.; Martinez, Eduardo; Boissard, Christopher G.; Post-Munson, Debra J.; Trojnacki, Joanne T.; Yeleswaram, Krishnaswamy; Pajor, Lorraine M.; Knipe, Jay; Gao, Qi; Perrone, Robert; Starrett, John E.Bioorganic & Medicinal Chemistry Letters (2002), 12 (7), 1023-1026CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science Ltd.)3-Aryl-3-fluorooxindoles can be efficiently synthesized in two steps by the addn. of an aryl Grignard to an isatin, followed by treatment with DAST. Oxindole 1 (BMS-204352; MaxiPost) can be isolated using chiral HPLC or prepd. by employing chiral resoln. Cloned maxi-K channels are opened by 1, which demonstrates a brain/plasma ratio >9 in rats.316Lee, K. L.; Ambler, C. M.; Anderson, D. R.; Boscoe, B. P.; Bree, A. G.; Brodfuehrer, J. I.; Chang, J. S.; Choi, C.; Chung, S.; Curran, K. J.; Day, J. E.; Dehnhardt, C. M.; Dower, K.; Drozda, S. E.; Frisbie, R. K.; Gavrin, L. K.; Goldberg, J. A.; Han, S.; Hegen, M.; Hepworth, D.; Hope, H. R.; Kamtekar, S.; Kilty, I. C.; Lee, A.; Lin, L. L.; Lovering, F. E.; Lowe, M. D.; Mathias, J. P.; Morgan, H. M.; Murphy, E. A.; Papaioannou, N.; Patny, A.; Pierce, B. S.; Rao, V. R.; Saiah, E.; Samardjiev, I. J.; Samas, B. M.; Shen, M. W. H.; Shin, J. H.; Soutter, H. H.; Strohbach, J. W.; Symanowicz, P. T.; Thomason, J. R.; Trzupek, J. D.; Vargas, R.; Vincent, F.; Yan, J.; Zapf, C. W.; Wright, S. W. Discovery of clinical candidate 1-{[(2S,3S,4S)-3-ethyl-4-fluoro-5-oxopyrrolidin-2-yl]methoxy}-7-methoxyisoquinoli ne-6-carboxamide (PF-06650833), a potent, selective inhibitor of interleukin-1 receptor associated kinase 4 (IRAK4), by fragment-based drug design. J. Med. Chem. 2017, 60, 5521– 5542, DOI: 10.1021/acs.jmedchem.7b00231[ACS Full Text], [CAS], Google Scholar316https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXnslGqu7c%253D&md5=9771ad2d33089f6ef3dba3556536ff1aDiscovery of Clinical Candidate 1-{[(2S,3S,4S)-3-Ethyl-4-fluoro-5-oxopyrrolidin-2-yl]methoxy}-7-methoxyisoquinoline-6-carboxamide (PF-06650833), a Potent, Selective Inhibitor of Interleukin-1 Receptor Associated Kinase 4 (IRAK4), by Fragment-Based Drug DesignLee, Katherine L.; Ambler, Catherine M.; Anderson, David R.; Boscoe, Brian P.; Bree, Andrea G.; Brodfuehrer, Joanne I.; Chang, Jeanne S.; Choi, Chulho; Chung, Seungwon; Curran, Kevin J.; Day, Jacqueline E.; Dehnhardt, Christoph M.; Dower, Ken; Drozda, Susan E.; Frisbie, Richard K.; Gavrin, Lori K.; Goldberg, Joel A.; Han, Seungil; Hegen, Martin; Hepworth, David; Hope, Heidi R.; Kamtekar, Satwik; Kilty, Iain C.; Lee, Arthur; Lin, Lih-Ling; Lovering, Frank E.; Lowe, Michael D.; Mathias, John P.; Morgan, Heidi M.; Murphy, Elizabeth A.; Papaioannou, Nikolaos; Patny, Akshay; Pierce, Betsy S.; Rao, Vikram R.; Saiah, Eddine; Samardjiev, Ivan J.; Samas, Brian M.; Shen, Marina W. H.; Shin, Julia H.; Soutter, Holly H.; Strohbach, Joseph W.; Symanowicz, Peter T.; Thomason, Jennifer R.; Trzupek, John D.; Vargas, Richard; Vincent, Fabien; Yan, Jiangli; Zapf, Christoph W.; Wright, Stephen W.Journal of Medicinal Chemistry (2017), 60 (13), 5521-5542CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Through fragment-based drug design focused on engaging the active site of IRAK4 and leveraging three-dimensional topol. in a ligand-efficient manner, a micromolar hit identified from a screen of a Pfizer fragment library was optimized to afford IRAK4 inhibitors with nanomolar potency in cellular assays. The medicinal chem. effort featured the judicious placement of lipophilicity, informed by cocrystal structures with IRAK4 and optimization of ADME properties to deliver clin. candidate I. This compd. benefitted from a 5-unit increase in lipophilic efficiency from the fragment hit, excellent kinase selectivity, and pharmacokinetic properties suitable for oral administration.317Dossetter, A. G. A statistical analysis of in vitro human microsomal metabolic stability of small phenyl group substituents, leading to improved design sets for parallel SAR exploration of a chemical series. Bioorg. Med. Chem. 2010, 18, 4405– 4414, DOI: 10.1016/j.bmc.2010.04.077[Crossref], [PubMed], [CAS], Google Scholar317https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXntV2rurc%253D&md5=93c9825d5da0b47e0f4c66996c883642A statistical analysis of in vitro human microsomal metabolic stability of small phenyl group substituents, leading to improved design sets for parallel SAR exploration of a chemical seriesDossetter, Alexander G.Bioorganic & Medicinal Chemistry (2010), 18 (12), 4405-4414CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)An anal. of in vitro human liver microsomal turnover assay results from a large dataset (∼75 K) of exptl. compds. tested is presented. Combined with an anal. of small (<6 Ha) substituents on known drugs and existing published results a new set of 29 substituents (consensus) is proposed to increase stability and probe SAR (an enhanced Topliss set'). In addn. a different group of 28 substituents are identified as unlikely to change in vitro HLM stability, and a further set of compds. focuses on increasing HLM stability only.318Lin, L. S.; Lanza, T. J., Jr.; Jewell, J. P.; Liu, P.; Shah, S. K.; Qi, H.; Tong, X.; Wang, J.; Xu, S. S.; Fong, T. M.; Shen, C. P.; Lao, J.; Xiao, J. C.; Shearman, L. P.; Stribling, D. S.; Rosko, K.; Strack, A.; Marsh, D. J.; Feng, Y.; Kumar, S.; Samuel, K.; Yin, W.; Van der Ploeg, L. H.; Goulet, M. T.; Hagmann, W. K. Discovery of N-[(1S,2S)-3-(4-Chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364), a novel, acyclic cannabinoid-1 receptor inverse agonist for the treatment of obesity. J. Med. Chem. 2006, 49, 7584– 7587, DOI: 10.1021/jm060996+[ACS Full Text], [CAS], Google Scholar318https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlCqsLvK&md5=e016bb35e491de4422bef3f3fa87ae84Discovery of N-[(1S,2S)-3-(4-Chlorophenyl)-2- (3-cyanophenyl)-1-methylpropyl]-2-methyl-2- {[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364), a Novel, Acyclic Cannabinoid-1 Receptor Inverse Agonist for the Treatment of ObesityLin, Linus S.; Lanza, Thomas J., Jr.; Jewell, James P.; Liu, Ping; Shah, Shrenik K.; Qi, Hongbo; Tong, Xinchun; Wang, Junying; Xu, Suoyu S.; Fong, Tung M.; Shen, Chun-Pyn; Lao, Julie; Xiao, Jing Chen; Shearman, Lauren P.; Stribling, D. Sloan; Rosko, Kimberly; Strack, Alison; Marsh, Donald J.; Feng, Yue; Kumar, Sanjeev; Samuel, Koppara; Yin, Wenji; Van der Ploeg, Lex H. T.; Goulet, Mark T.; Hagmann, William K.Journal of Medicinal Chemistry (2006), 49 (26), 7584-7587CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The discovery of novel acyclic amide cannabinoid-1 receptor inverse agonists is described. They are potent, selective, orally bioavailable, and active in rodent models of food intake and body wt. redn. A major focus of the optimization process was to increase in vivo efficacy and to reduce the potential for formation of reactive metabolites. These efforts led to the identification of compd. 48 (I)for development as a clin. candidate for the treatment of obesity.319Elliott, E. C.; Regan, S. L.; Maggs, J. L.; Bowkett, E. R.; Parry, L. J.; Williams, D. P.; Park, B. K.; Stachulski, A. V. Haloarene derivatives of carbamazepine with reduced bioactivation liabilities: 2-monohalo and 2,8-dihalo derivatives. J. Med. Chem. 2012, 55, 9773– 9784, DOI: 10.1021/jm301013n[ACS Full Text], [CAS], Google Scholar319https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFCrur7J&md5=d158092424146266ca119a4d5656ba30Haloarene Derivatives of Carbamazepine with Reduced Bioactivation Liabilities: 2-Monohalo and 2,8-Dihalo DerivativesElliott, Emma-Claire; Regan, Sophie L.; Maggs, James L.; Bowkett, Elizabeth R.; Parry, Laura J.; Williams, Dominic P.; Park, B. Kevin; Stachulski, Andrew V.Journal of Medicinal Chemistry (2012), 55 (22), 9773-9784CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The anticonvulsant carbamazepine (I) is assocd. with adverse drug reactions (ADRs), including hepatotoxicity; oxidative metab. of I has been implicated in the pathogenesis of the ADRs. The authors report the synthesis and evaluation of 2-monohalo and 2,8-dihalo analogs of I that were intended to minimize reactive metabolite formation via arene oxidn. and 10,11-epoxidn. Halo analogs were obtained either by rearrangement of halogenated N-arylindoles or from specifically halogenated iminodibenzyl derivs. In rat hepatocytes, none of the analogs underwent oxidative dehalogenation or glutathione adduction. Some formation of the 10,11-epoxide still occurred, but arom. hydroxylation was not seen with the exception of 2-fluoro, which allowed minor monohydroxylation. Complete inhibition of arom. hydroxylation required at least monochlorination or difluorination of I. In human liver microsomes, difluoro analog II underwent 10,11-epoxidn. but gave no arene oxidn.320Hansch, C.; Leo, A.; Unger, S. H.; Kim, K. H.; Nikaitani, D.; Lien, E. J. ″Aromatic″ substituent constants for structure-activity correlations. J. Med. Chem. 1973, 16, 1207– 1216, DOI: 10.1021/jm00269a003[ACS Full Text], [CAS], Google Scholar320https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3sXlt1Wkt7o%253D&md5=e0ee110086f19b9853838f47ba1c1d4dAromatic substituent constants for structure-activity correlationsHansch, Corwin; Leo, Albert; Unger, Stefan H.; Kim, Ki Hwai; Nikaitani, Donald; Lien, Eric J.Journal of Medicinal Chemistry (1973), 16 (11), 1207-16CODEN: JMCMAR; ISSN:0022-2623.The arom. substituent consts. π (lipophilic), σm and σp (electronic), and molar refractivity (steric) were collected from the literature for 236 substituents, including 128 π values and 191 values for which both σm and σp were found. The field and resonance electronic properties of C. G. Swain and E. C. Lupton (1968) were calcd. for these 191 substituents by a corrected procedure and were essentially orthogonal. The mutual correlation of σm and σp was high.321Samuel, K.; Yin, W.; Stearns, R. A.; Tang, Y. S.; Chaudhary, A. G.; Jewell, J. P.; Lanza, T., Jr.; Lin, L. S.; Hagmann, W. K.; Evans, D. C.; Kumar, S. Addressing the metabolic activation potential of new leads in drug discovery: a case study using ion trap mass spectrometry and tritium labeling techniques. J. Mass Spectrom. 2003, 38, 211– 221, DOI: 10.1002/jms.434[Crossref], [PubMed], [CAS], Google Scholar321https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhsFGmurc%253D&md5=219eb210acef4357bfba39aec0576414Addressing the metabolic activation potential of new leads in drug discovery: A case study using ion trap mass spectrometry and tritium labeling techniquesSamuel, Koppara; Yin, Wenji; Stearns, Ralph A.; Tang, Yui S.; Chaudhary, Ashok G.; Jewell, James P.; Lanza, Thomas, Jr.; Lin, Linus S.; Hagmann, William K.; Evans, David C.; Kumar, SanjeevJournal of Mass Spectrometry (2003), 38 (2), 211-221CODEN: JMSPFJ; ISSN:1076-5174. (John Wiley & Sons Ltd.)Metabolic activation of drug candidates to electrophilic reactive metabolites that can covalently modify cellular macromols. may result in acute and/or idiosyncratic immune system-mediated toxicities in humans. This presents a significant potential liability for the future development of these compds. as safe therapeutic agents. We present here an example of an approach where sites of metabolic activation within a new drug candidate series were rapidly identified using online liq. chromatog./multistage mass spectrometry on an ion trap mass spectrometer. This was accomplished by trapping the reactive intermediates formed upon incubation of compds. with rat and human liver microsomes as their corresponding glutathione conjugates and mass spectral characterization of these thiol adducts. Based on the structures of the GSH adducts identified, potential sites and mechanisms of bioactivation within the chem. structure were proposed. These metab. studies were interfaced with iterative structural modifications of the chem. series in order to block these bioactivation sites within the mol. This strategy led to a significant redn. in the propensity of the compds. to undergo metabolic activation as evidenced by redns. in the irreversible binding of radioactivity to liver microsomal material upon incubation of tritium-labeled compds. with this in vitro system. With the efficiency and throughput achievable with such an approach, it appears feasible to identify and address the metabolic activation potential of new drug leads during routine metabolite identification studies in an early drug discovery setting.322Koerts, J.; Soffers, A. E.; Vervoort, J.; De Jager, A.; Rietjens, I. M. Occurrence of the NIH shift upon the cytochrome P450-catalyzed in vivo and in vitro aromatic ring hydroxylation of fluorobenzenes. Chem. Res. Toxicol. 1998, 11, 503– 512, DOI: 10.1021/tx980053i[ACS Full Text], [CAS], Google Scholar322https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXjt1Giurc%253D&md5=64178da1e0f25584e5aa883b57d3498cOccurrence of the NIH Shift upon the Cytochrome P450-Catalyzed in Vivo and in Vitro Aromatic Ring Hydroxylation of FluorobenzenesKoerts, Janneke; Soffers, Ans E. M. F.; Vervoort, Jacques; De Jager, Adrie; Rietjens, Ivonne M. C. M.Chemical Research in Toxicology (1998), 11 (5), 503-512CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)The in vivo cytochrome P 450-catalyzed arom. hydroxylation of a series of fluorobenzenes was investigated with special emphasis on the importance of the fluorine NIH shift. The results obtained demonstrate a minor role for the NIH shift in the metab. of the fluorobenzenes to phenolic metabolites in control male Wistar rats. These in vivo results could indicate that (1) the NIH shift is an inherently minor process for fluorine substituents or (2) it is a potentially significant process but the presumed epoxide that leads to formation of the NIH-shifted metabolite is lost to an alternative metabolic pathway. In contrast to the in vivo data, in vitro expts. showed a significant amt. of NIH-shifted metabolites for 1,4-difluorobenzene. This result eliminates the explanation that the NIH shift is an inherently minor process for fluorine substituents. Results of addnl. expts. presented in this paper show that the reduced tendency of fluorine-substituted benzenes to undergo an NIH shift in vivo can-at least in part-be ascribed to the possible existence of alternative pathways for metab. of the epoxide, such as, for example, GSH conjugation, being more efficient for fluorinated than chlorinated benzenes.323Dear, G. J.; Ismail, I. M.; Mutch, P. J.; Plumb, R. S.; Davies, L. H.; Sweatman, B. C. Urinary metabolites of a novel quinoxaline non-nucleoside reverse transcriptase inhibitor in rabbit, mouse and human: identification of fluorine NIH shift metabolites using NMR and tandem MS. Xenobiotica 2000, 30, 407– 426, DOI: 10.1080/004982500237604[Crossref], [PubMed], [CAS], Google Scholar323https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXmtFygtbk%253D&md5=ea3b3a235386d9664d5365a5929ed838Urinary metabolites of a novel quinoxaline non-nucleoside reverse transcriptase inhibitor in rabbit, mouse and human: identification of fluorine NIH shift metabolites using NMR and tandem MSDear, G. J.; Ismail, I. M.; Mutch, P. J.; Plumb, R. S.; Davies, L. H.; Sweatman, B. C.Xenobiotica (2000), 30 (4), 407-426CODEN: XENOBH; ISSN:0049-8254. (Taylor & Francis Ltd.)1. The urinary metabolites of (S)-2-ethyl-7-fluoro-3-oxo-3,4-dihydro-2H-quinoxaline-carboxylic acid isopropylester (GW420867X) have been investigated in samples obtained following oral administration to rabbit, mouse and human. GW420867X underwent extensive biotransformation to form hydroxylated metabolites and glucuronide conjugates on the arom. ring, and on the Et and iso-Pr side-chains in all species. In rabbit urine, a minor metabolite was detected and characterized as a cysteine adduct that was not obsd. in mouse or man. 2. The hydroxylated metabolites and corresponding glucuronide conjugates were isolated by semi-preparative HPLC and characterized using NMR, LC-NMR and LC-MS/MS. The relative proportions of fluorine-contg. metabolites were detd. in animal species by 19F-NMR signal integration. 3. The fluorine atom of the arom. ring underwent NIH shift rearrangement in the metabolites isolated and characterized in rabbit, mouse and human urine. 4. The characterization of the NIH shift metabolites in urine enabled the detection and confirmation of the presence of these metabolites in human plasma.324Gunduz, M.; Argikar, U. A.; Kamel, A.; Colizza, K.; Bushee, J. L.; Cirello, A.; Lombardo, F.; Harriman, S. Oxidative ipso substitution of 2,4-difluoro-benzylphthalazines: identification of a rare stable quinone methide and subsequent GSH conjugate. Drug Metab. Dispos. 2012, 40, 2074– 2080, DOI: 10.1124/dmd.112.046268[Crossref], [PubMed], [CAS], Google Scholar324https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFKkt73J&md5=228931ded8e0cae39915b3f0a2f694b0Oxidative ipso substitution of 2,4-difluoro-benzylphthalazines: identification of a rare stable quinone methide and subsequent GSH conjugateGunduz, Mithat; Argikar, Upendra A.; Kamel, Amin; Colizza, Kevin; Bushee, Jennifer L.; Cirello, Amanda; Lombardo, Franco; Harriman, ShawnDrug Metabolism & Disposition (2012), 40 (11), 2074-2080CODEN: DMDSAI; ISSN:1521-009X. (American Society for Pharmacology and Experimental Therapeutics)In vitro metabolite identification and GSH trapping studies in human liver microsomes were conducted to understand the bioactivation potential of compd. 1 [2-(6-(4-(4-(2,4-difluorobenzyl)phthalazin-1-yl)piperazin-1-yl)pyridin-3-yl)propan-2-ol], an inhibitor of the Hedgehog pathway. The results revealed the formation of a unique, stable quinone methide metabolite (M1) via ipso substitution of a fluorine atom and subsequent formation of a GSH adduct (M2). The stability of this metabolite arises from extensive resonance-stabilized conjugation of the substituted benzylphthalazine moiety. Cytochrome P 450 (P 450) phenotyping studies revealed that the formation of M1 and M2 were NADPH-dependent and primarily catalyzed by CYP3A4 among the studied P 450 isoforms. In summary, an unusual and stable quinone methide metabolite of compd. 1 was identified, and a mechanism was proposed for its formation via an oxidative ipso substitution.325Chen, H.; Shockcor, J.; Chen, W.; Espina, R.; Gan, L. S.; Mutlib, A. E. Delineating novel metabolic pathways of DPC 963, a non-nucleoside reverse transcriptase inhibitor, in rats. Characterization of glutathione conjugates of postulated oxirene and benzoquinone imine intermediates by LC/MS and LC/NMR. Chem. Res. Toxicol. 2002, 15, 388– 399, DOI: 10.1021/tx010153f[ACS Full Text], [CAS], Google Scholar325https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XhtFalu7o%253D&md5=ff34e05f9eb59f8ff07ef2698433d166Delineating novel metabolic pathways of DPC 963, a non-nucleoside reverse transcriptase inhibitor, in rats. Characterization of glutathione conjugates of postulated oxirene and benzoquinone imine intermediates by LC/MS and LC/NMRChen, Hao; Shockcor, John; Chen, Weiqi; Espina, Robert; Gan, Liang-Shang; Mutlib, A. E.Chemical Research in Toxicology (2002), 15 (3), 388-399CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)The metabolic activation of (S)-5,6-difluoro-4-cyclopropylethynyl-4-trifluoromethyl-3,4-dihydro-2(1H)-quinazolinone, DPC 963, in rats was investigated by identifying and characterizing the GSH and mercapturic acid conjugates excreted in the bile and urine, resp. The structures of these adducts, which were unequivocally elucidated by LC/MS/MS and NMR expts., revealed the existence of at least three distinct metabolic pathways leading to these products. One of the pathways, which has been described previously, involves the activation of the acetylene group after an initial hydroxylation on the methine carbon of the cyclopropyl ring. Metabolite M1 was demonstrated to be formed via this pathway after an enzymic addn. of GSH across the triple bond of the substituted acetylene. The second pathway, also previously described, leads to diastereoisomeric GSH adducts M3 and M4 after the formation of a highly reactive oxirene intermediate. This postulated oxirene subsequently rearranges to an α, β-unsatd. cyclobutenyl ketone intermediate capable of undergoing a 1,4-Michael addn. with a nucleophile such as GSH. In addn. to these pathways, DPC 963 was found to undergo a metabolic activation previously undescribed for structural analogs of this compd. It is postulated that an oxidative defluorination mediated by cytochrome P 450 leads to the formation of a putative benzoquinone imine intermediate which subsequently reacts with GSH to form two arom. ring-substituted regioisomeric conjugates, M5 and M6. In addn. to forming the GSH adducts, the benzoquinone imine was also found to be reduced to its unreactive hydroquinone metabolite, which was excreted as the glucuronide conjugate in rat bile. Studies with induced rat microsomes, cDNA-expressed rat P 450 isoenzymes, and polyclonal antibodies against rat P 450 clearly demonstrated that the rat P450s 3A1/3A2 were responsible for the formation of postulated oxirene and benzoquinone intermediates.326Hebner, C. M.; Han, B.; Brendza, K. M.; Nash, M.; Sulfab, M.; Tian, Y.; Hung, M.; Fung, W.; Vivian, R. W.; Trenkle, J.; Taylor, J.; Bjornson, K.; Bondy, S.; Liu, X.; Link, J.; Neyts, J.; Sakowicz, R.; Zhong, W.; Tang, H.; Schmitz, U. The HCV non-nucleoside inhibitor Tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase function. PLoS One 2012, 7, e39163 DOI: 10.1371/journal.pone.0039163[Crossref], [PubMed], [CAS], Google Scholar326https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XovF2jtro%253D&md5=55998f37569ff6b657425bf95a0b677dThe HCV non-nucleoside inhibitor tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase functionHebner, Christy M.; Han, Bin; Brendza, Katherine M.; Nash, Michelle; Sulfab, Maisoun; Tian, Yang; Hung, Magdeleine; Fung, Wanchi; Vivian, Randall W.; Trenkle, James; Taylor, James; Bjornson, Kyla; Bondy, Steven; Liu, Xiaohong; Link, John; Neyts, Johan; Sakowicz, Roman; Zhong, Weidong; Tang, Hengli; Schmitz, UliPLoS One (2012), 7 (6), e39163CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Tegobuvir (TGV) is a novel non-nucleoside inhibitor (NNI) of HCV RNA replication with demonstrated antiviral activity in patients with genotype 1 chronic HCV infection. The mechanism of action of TGV has not been clearly defined despite the identification of resistance mutations mapping to the NS5B polymerase region. TGV does not inhibit NS5B enzymic activity in biochem. assays in vitro, suggesting a more complex antiviral mechanism with cellular components. Here, we demonstrate that TGV exerts anti-HCV activity utilizing a unique chem. activation and subsequent direct interaction with the NS5B protein. Treatment of HCV subgenomic replicon cells with TGV results in a modified form of NS5B with a distinctly altered mobility on a SDS-PAGE gel. Further anal. reveals that the aberrantly migrating NS5B species contains the inhibitor mol. Formation of this complex does not require the presence of any other HCV proteins. The intensity of the aberrantly migrating NS5B species is strongly dependent on cellular glutathione levels as well as CYP 1A activity. Furthermore anal. of NS5B protein purified from a heterologous expression system treated with TGV by mass spectrometry suggests that TGV undergoes a CYP-mediated intracellular activation step and the resulting metabolite, after forming a glutathione conjugate, directly and specifically interacts with NS5B. Taken together, these data demonstrate that upon metabolic activation TGV is a specific, covalent inhibitor of the HCV NS5B polymerase and is mechanistically distinct from other classes of the non-nucleoside inhibitors (NNI) of the viral polymerase.327Powers, J. P.; Piper, D. E.; Li, Y.; Mayorga, V.; Anzola, J.; Chen, J. M.; Jaen, J. C.; Lee, G.; Liu, J.; Peterson, M. G.; Tonn, G. R.; Ye, Q.; Walker, N. P.; Wang, Z. SAR and mode of action of novel non-nucleoside inhibitors of hepatitis C NS5b RNA polymerase. J. Med. Chem. 2006, 49, 1034– 1046, DOI: 10.1021/jm050859x[ACS Full Text], [CAS], Google Scholar327https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XksV2ltA%253D%253D&md5=722ac0edb5dabb720e30475ef5925d5fSAR and Mode of Action of Novel Non-Nucleoside Inhibitors of Hepatitis C NS5b RNA PolymerasePowers, Jay P.; Piper, Derek E.; Li, Yang; Mayorga, Veronica; Anzola, John; Chen, James M.; Jaen, Juan C.; Lee, Gary; Liu, Jinqian; Peterson, M. Greg; Tonn, George R.; Ye, Qiuping; Walker, Nigel P. C.; Wang, ZhulunJournal of Medicinal Chemistry (2006), 49 (3), 1034-1046CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Novel non-nucleoside inhibitors of the HCV RNA polymerase (NS5b) with sub-micromolar biochem. potency have been identified which are selective for the inhibition of HCV NS5b over other polymerases. The structures of the complexes formed between several of these inhibitors and HCV NS5b were detd. by x-ray crystallog., and the inhibitors were found to bind in an allosteric binding site sep. from the active site. Structure-activity relationships and structural studies have identified the mechanism of action for compds. in this series, several of which possess drug-like properties, as unique, reversible, covalent inhibitors of HCV NS5b.328Mandal, M.; Mitra, K.; Grotz, D.; Lin, X.; Palamanda, J.; Kumari, P.; Buevich, A.; Caldwell, J. P.; Chen, X.; Cox, K.; Favreau, L.; Hyde, L.; Kennedy, M. E.; Kuvelkar, R.; Liu, X.; Mazzola, R. D.; Parker, E.; Rindgen, D.; Sherer, E.; Wang, H.; Zhu, Z.; Stamford, A. W.; Cumming, J. N. Overcoming time-dependent inhibition (TDI) of cytochrome P450 3A4 (CYP3A4) resulting from bioactivation of a fluoropyrimidine moiety. J. Med. Chem. 2018, 61, 10700– 10708, DOI: 10.1021/acs.jmedchem.8b01326[ACS Full Text], [CAS], Google Scholar328https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitV2rtbfM&md5=09c862a66c3b078e0886696a6c2cd489Overcoming Time-Dependent Inhibition (TDI) of Cytochrome P450 3A4 (CYP3A4) Resulting from Bioactivation of a Fluoropyrimidine MoietyMandal, Mihirbaran; Mitra, Kaushik; Grotz, Diane; Lin, Xinjie; Palamanda, Jairam; Kumari, Pramila; Buevich, Alexei; Caldwell, John P.; Chen, Xia; Cox, Kathleen; Favreau, Leonard; Hyde, Lynn; Kennedy, Matthew E.; Kuvelkar, Reshma; Liu, Xiaoxiang; Mazzola, Robert D.; Parker, Eric; Rindgen, Diane; Sherer, Edward; Wang, Hongwu; Zhu, Zhaoning; Stamford, Andrew W.; Cumming, Jared N.Journal of Medicinal Chemistry (2018), 61 (23), 10700-10708CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Herein the authors describe structure-activity relationship (SAR) and metabolite identification (Met-ID) studies that provided insight into the origin of time-dependent inhibition (TDI) of cytochrome P 450 3A4 (CYP3A4) by compd. I. Collectively, these efforts revealed that bioactivation of the fluoropyrimidine moiety of I led to reactive metabolite formation via oxidative defluorination and was responsible for the obsd. TDI. The authors discovered that substitution at both the 4- and 6-positions of the 5-fluoropyrimidine of I was necessary to ameliorate this TDI as exemplified by compd. II.329Wermers, R. A.; Cooper, K.; Razonable, R. R.; Deziel, P. J.; Whitford, G. M.; Kremers, W. K.; Moyer, T. P. Fluoride excess and periostitis in transplant patients receiving long-term voriconazole therapy. Clin. Infect. Dis. 2011, 52, 604– 611, DOI: 10.1093/cid/ciq188[Crossref], [PubMed], [CAS], Google Scholar329https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhslWmur8%253D&md5=d9af38baae224a2a97d87a8d6693a10dFluoride Excess and Periostitis in Transplant Patients Receiving Long-Term Voriconazole TherapyWermers, Robert A.; Cooper, Kay; Razonable, Raymund R.; Deziel, Paul J.; Whitford, Gary M.; Kremers, Walter K.; Moyer, Thomas P.Clinical Infectious Diseases (2011), 52 (5), 604-611CODEN: CIDIEL; ISSN:1058-4838. (Oxford University Press)We have identified that in transplant subjects, long term use of voriconazole, a fluoride contg. medication, is assocd. with elevated plasma fluoride levels compared to those not taking it. Fluoride related bone complications were also obsd. including periostitis and fluorosis. We describe a heart transplant patient with painful periostitis and exostoses who was receiving long-term therapy with voriconazole, which is a fluoride-contg. medication. Elevated plasma and bone fluoride levels were identified. Discontinuation of voriconazole therapy led to improvement in pain and reduced fluoride and alk. phosphatase levels. To det. whether voriconazole is a cause of fluoride excess, we measured plasma fluoride levels in 10 adult post-transplant patients who had received voriconazole for at least 6 mo and 10 post-transplant patients who did not receive voriconazole. To assess the effect of renal insufficiency on fluoride levels in subjects receiving voriconazole, half were recruited on the basis of a serum creatinine level of 1.4 mg/dL on their most recent measurement, whereas the other 5 subjects receiving voriconazole had serum creatinine levels <1.4 mg/dL. All control subjects had serum creatinine levels of 1.4 mg/dL. Patients were excluded from the study if they received a fluorinated pharmaceutical other than voriconazole. All subjects who received voriconazole had elevated plasma fluoride levels, and no subjects in the control group had elevated levels (14.32 μmol/L ± 6.41 vs 2.54 ± 0.67 μmol/L; P<.001). Renal function was not predictive of fluoride levels. Plasma fluoride levels remained significantly higher in the voriconazole group after adjusting for calcineurin inhibitor levels and doses. Half of the voriconazole group subjects had evidence of periostitis, including exostoses in 2 patients. Discontinuation of voriconazole therapy in patients with periostitis resulted in improvement of pain and a redn. in alk. phosphatase and fluoride levels. Voriconazole is assocd. with painful periostitis, exostoses, and fluoride excess in post-transplant patients with long-term voriconazole use.330Wang, T. F.; Wang, T.; Altman, R.; Eshaghian, P.; Lynch, J. P., 3rd; Ross, D. J.; Belperio, J. A.; Weigt, S. S.; Saggar, R.; Gregson, A.; Kubak, B.; Saggar, R. Periostitis secondary to prolonged voriconazole therapy in lung transplant recipients. Am. J. Transplant. 2009, 9, 2845– 2850, DOI: 10.1111/j.1600-6143.2009.02837.x[Crossref], [PubMed], [CAS], Google Scholar330https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1MfjtlCitw%253D%253D&md5=df2416a750f142fcda08d0a6c460b5c7Periostitis secondary to prolonged voriconazole therapy in lung transplant recipientsWang T F; Wang T; Altman R; Eshaghian P; Lynch J P 3rd; Ross D J; Belperio J A; Weigt S S; Saggar R; Gregson A; Kubak B; Saggar RAmerican journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons (2009), 9 (12), 2845-50 ISSN:.We report five cases of possible drug-induced periostitis associated with long-term use of voriconazole therapy after lung transplantation (LT). The diagnosis of periostitis was made by the documentation of bone pain, elevation of serum alkaline phosphatase and characteristic findings on radionuclide bone imaging in the absence of any identifiable rheumatologic disease. This periostitis appears similar to hypertrophic osteoarthopathy (HOA) but does not meet all criteria for HOA. In all patients, the symptoms resolved rapidly after discontinuation of voriconazole therapy. Awareness of this potential syndrome, which manifests as bone pain, elevated serum alkaline phosphatase and a bone scan suggestive of periostitis, is necessary in LT recipients on long-term voriconazole.331Chen, L.; Mulligan, M. E. Medication-induced periostitis in lung transplant patients: periostitis deformans revisited. Skeletal Radiol 2011, 40, 143– 148, DOI: 10.1007/s00256-010-0997-y[Crossref], [PubMed], [CAS], Google Scholar331https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3M%252Fmt1OnsQ%253D%253D&md5=d44b813266dcdbeb57fe550c341ab117Medication-induced periostitis in lung transplant patients: periostitis deformans revisitedChen Lina; Mulligan Michael ESkeletal radiology (2011), 40 (2), 143-8 ISSN:.We report five cases of diffuse periostitis resembling hypertrophic osteoarthropathy and perostitis deformans in lung transplantation patients on chronic voriconazole, a fluoride-containing compound. Although drug-related periostitis has long been known, the association of lung transplant medication with periostitis was only recently introduced in the literature. To our knowledge, imaging findings have not been fully characterized in the radiology literature. Imaging features along with clinical history help to distinguish this benign condition from other disease entities. In this article, we review the current literature and illustrate the variety of imaging characteristics of this entity so that interpreting radiologists can make accurate diagnoses and avoid unnecessary work up.332Roffey, S. J.; Cole, S.; Comby, P.; Gibson, D.; Jezequel, S. G.; Nedderman, A. N.; Smith, D. A.; Walker, D. K.; Wood, N. The disposition of voriconazole in mouse, rat, rabbit, guinea pig, dog, and human. Drug Metab. Dispos. 2003, 31, 731– 741, DOI: 10.1124/dmd.31.6.731[Crossref], [PubMed], [CAS], Google Scholar332https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXkt1KrtLw%253D&md5=831768b6c72bce3a8528af680c60a3d9The disposition of voriconazole in mouse, rat, rabbit, guinea pig, dog, and humanRoffey, S. J.; Cole, S.; Comby, P.; Gibson, D.; Jezequel, S. G.; Nedderman, A. N. R.; Smith, D. A.; Walker, D. K.; Wood, N.Drug Metabolism and Disposition (2003), 31 (6), 731-741CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)Voriconazole is a new triazole antifungal agent with potent, wide-spectrum activity. Its pharmacokinetics and metab. have been studied in mouse, rat, rabbit, dog, guinea pig, and humans after single and multiple administration by both oral and i.v. routes. Absorption of voriconazole is essentially complete in all species. The elimination of voriconazole is characterized by non-linear pharmacokinetics in all species. Consequently, pharmacokinetic parameters are dependent upon dose, and a superproportional increase in area under the curve is seen with increasing dose in rat and dog toxicol. studies. Following multiple administration, there is a decrease in systemic exposure. This is most pronounced in mouse and rat, less so in dog, and not obsd. in guinea pig or rabbit. Repeat-dose toxicol. studies in mouse, rat, and dog have demonstrated that induction of cytochrome P 450 by voriconazole (autoinduction of metab.) is responsible for the decreased exposure in these species. Autoinduction of metab. is not obsd. in humans, and plasma steady-state concns. remain const. with time. Voriconazole is extensively metabolized in all species. The major pathways in humans involve fluoropyrimidine N-oxidn., fluoropyrimidine hydroxylation, and Me hydroxylation. Also, N-oxidn. facilitates cleavage of the mol., resulting in loss of the fluoropyrimidine moiety and subsequent conjugation with glucuronic acid. Major pathways are represented in animal species. The major circulating metabolite in rat, dog, and human is the N-oxide of voriconazole. It is not thought to contribute to efficacy since it is at least 100-fold less potent than voriconazole against fungal pathogens in vitro.333Schulz, J.; Kluwe, F.; Mikus, G.; Michelet, R.; Kloft, C. Novel insights into the complex pharmacokinetics of voriconazole: a review of its metabolism. Drug Metab. Rev. 2019, 51, 247– 265, DOI: 10.1080/03602532.2019.1632888[Crossref], [PubMed], [CAS], Google Scholar333https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsF2lsrvP&md5=b6209461b8176d373d14b3f90205f69eNovel insights into the complex pharmacokinetics of voriconazole: a review of its metabolismSchulz, Josefine; Kluwe, Franziska; Mikus, Gerd; Michelet, Robin; Kloft, CharlotteDrug Metabolism Reviews (2019), 51 (3), 247-265CODEN: DMTRAR; ISSN:0360-2532. (Taylor & Francis Ltd.)A review. Voriconazole, a second-generation triazole frequently used for the prophylaxis and treatment of invasive fungal infections, undergoes complex metab. mainly involving various (polymorphic) cytochrome P 450 enzymes in humans. Although high inter- and intraindividual variability in voriconazole pharmacokinetics have been obsd. and the therapeutic range for this compd. is relatively narrow, the metab. of voriconazole has not been fully elucidated yet. The available literature data investigating the multiple different pathways and metabolites areextremely unbalanced and thus the abs. or relative contribution of the different pathwaysand enzymes involved in the metab. of voriconazole remains uncertain. Furthermore, other factors such as nonlinear pharmacokinetics caused by auto-inhibition or -induction and polymorphisms of the metabolizing enzymes hinder safe and effective voriconazole dosing in clin. practice and have not yet been studied sufficiently. This review aimed at amalgamating the available literature on the pharmacokinetics of voriconazole in vitro and in vivo, with a special focuson metab. in adults and children, in order to congregate an overall landscape of the currentbody of knowledge and identify knowledge gaps, opening the way towards further research inorder to foster the understanding, towards better therapeutic dosing decisions.334Shang, J.; Xu, S.; Teffera, Y.; Doss, G. A.; Stearns, R. A.; Edmonson, S.; Beconi, M. G. Metabolic activation of a pentafluorophenylethylamine derivative: formation of glutathione conjugates in vitro in the rat. Xenobiotica 2005, 35, 697– 713, DOI: 10.1080/00498250500230479[Crossref], [PubMed], [CAS], Google Scholar334https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1Sns73K&md5=17f4ddc8b005c5ad153497adbba25524Metabolic activation of a pentafluorophenylethylamine derivative: Formation of glutathione conjugates in vitro in the ratShang, J.; Xu, S.; Teffera, Y.; Doss, G. A.; Stearns, R. A.; Edmonson, S.; Beconi, M. G.Xenobiotica (2005), 35 (7), 697-713CODEN: XENOBH; ISSN:0049-8254. (Taylor & Francis Ltd.)The aim was to investigate the metabolic activation potential of a pentafluorophenylethylamine deriv. (compd. I) in vitro in the rat and to identify the cytochrome P 450 (CYP) enzymes that catalyze these metabolic activation processes. Reduced glutathione (GSH) was fortified in rat hepatocytes and liver microsomes to trap possible reactive intermediates. Four glutathione conjugates (M1-4) were identified by LC-MSn following incubation of compd. I in GSH-enriched rat hepatocytes and liver microsomes. Three of these conjugates (M2-4) have not been reported previously for pentafluorophenyl derivs. Elemental compn. anal. of these conjugates was obtained using high-resoln. quadrupole time-of-flight mass spectrometry. The formation of GSH conjugate M1 was rationalized as a direct nucleophilic replacement of fluoride by glutathione, whereas the formation of the GSH conjugates M2-4 was proposed to occur by NADPH-dependent metabolic activation of the pentafluorophenyl ring via arene oxide, quinone and/or quinoneimine reactive intermediates. Formation of these conjugates was enhanced three- to five-fold in liver microsomes obtained from phenobarbital- and dexamethasone-treated rats. In incubations with pooled rat liver microsomes and recombinant rat CYP3A1 and CYP3A2, troleandomycin (TAO) reduced the formation of GSH conjugates M2-4 by 80 - 90%, but it had no effect on the formation of M1. Incubation of compd. I with rat supersomes indicated that only CYP3A1 and CYP3A2 were capable of mediating these metabolic activation processes.335Shan, B.; Medina, J. C.; Santha, E.; Frankmoelle, W. P.; Chou, T. C.; Learned, R. M.; Narbut, M. R.; Stott, D.; Wu, P.; Jaen, J. C.; Rosen, T.; Timmermans, P. B.; Beckmann, H. Selective, covalent modification of β-tubulin residue Cys-239 by T138067, an antitumor agent with in vivo efficacy against multidrug-resistant tumors. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 5686– 5691, DOI: 10.1073/pnas.96.10.5686[Crossref], [PubMed], [CAS], Google Scholar335https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXjtFCntLY%253D&md5=3dd9933e4db4cbb8875e7213c8e86ad8Selective, covalent modification of β-tubulin residue Cys-239 by T138067, an antitumor agent with in vivo efficacy against multidrug-resistant tumorsShan, Bei; Medina, Julio C.; Santha, Edit; Frankmoelle, Walter P.; Chou, Ting-C.; Learned, Robert M.; Narbut, Mathew R.; Stott, Dean; Wu, Pengguang; Jaen, Juan C.; Rosen, Terry; Timmermans, Pieter B. M. W. M.; Beckmann, HolgerProceedings of the National Academy of Sciences of the United States of America (1999), 96 (10), 5686-5691CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Microtubules are linear polymers of α- and β-tubulin heterodimers and are the major constituents of mitotic spindles, which are essential for the sepn. of chromosomes during mitosis. Here we describe a synthetic compd., 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene (T138067), which covalently and selectively modifies the β1, β2, and β4 isotypes of β-tubulin at a conserved cysteine residue, thereby disrupting microtubule polymn. Cells exposed to T138067 become altered in shape, indicating a collapse of the cytoskeleton, and show an increase in chromosomal ploidy. Subsequently, these cells undergo apoptosis. Furthermore, T138067 exhibits cytotoxicity against tumor cell lines that exhibit substantial resistance to vinblastine, paclitaxel, doxorubicin, and actinomycin D. T138067 is also equally efficacious in inhibiting the growth of sensitive and multidrug-resistant human tumor xenografts in athymic nude mice. These observations suggest that T138067 may be clin. useful for the treatment of multidrug-resistant tumors.336Frankmoelle, W. P.; Medina, J. C.; Shan, B.; Narbut, M. R.; Beckmann, H. Glutathione S-transferase metabolism of the antineoplastic pentafluorophenylsulfonamide in tissue culture and mice. Drug Metab. Dispos. 2000, 28, 951– 958[PubMed], [CAS], Google Scholar336https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXlt1aht7w%253D&md5=8045e24a133a8c1d754df9c9d45cb665Glutathione S-transferase metabolism of the antineoplastic pentafluorophenylsulfonamide in tissue culture and miceFrankmoelle, Walter P.; Medina, Julio C.; Shan, Bei; Narbut, Mathew R.; Beckmann, HolgerDrug Metabolism and Disposition (2000), 28 (8), 951-958CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)The microtubule disrupting agent 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene (T138067) binds covalently and selectively to β-tubulin and has been shown to evade drug-efflux pumps that confer multidrug resistance to other antimitotic drugs that are used in cancer chemotherapy. In addn. to these resistance mechanisms, eukaryotic cells have developed other protection mechanisms that involve enzymes that modify electrophilic xenobiotics. To det. whether T138067 is a substrate for such enzymic detoxification pathways, a metab. study was initiated. GSH conjugation was shown to play a major role in T138067 metab. T138067-GSH conjugates were isolated from the culture media of T138067-treated cells and the bile of mice treated i.v. with T138067. The major T138067-GSH degrdn. products were also isolated from these sources. 19F NMR studies of the metabolites showed that metabolic conversions occurred through substitution of the para fluorine atom in the pentafluorophenyl ring of T138067. The T138067-GSH conjugate was also isolated from T138067 incubation buffer that had been exposed to mouse, rat, dog, or human liver slices, suggesting that this mechanism is not species-specific. All three human glutathione S-transferases (α, μ, and π), which are expressed in a wide variety of tissues including human tumors, were shown to metabolize T138067 effectively in vitro. The combined data show that T138067 is being metabolized, in vitro and in vivo, predominantly via a glutathione S-transferase-mediated metabolic pathway.337Yang, J.; Li, Y.; Yan, W.; Li, W.; Qiu, Q.; Ye, H.; Chen, L. Covalent modification of Cys-239 in β-tubulin by small molecules as a strategy to promote tubulin heterodimer degradation. J. Biol. Chem. 2019, 294, 8161– 8170, DOI: 10.1074/jbc.RA118.006325[Crossref], [PubMed], [CAS], Google Scholar337https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtFOnurfP&md5=e92427e7fe735c00f366af467eafe233Covalent modification of Cys-239 in β-tubulin by small molecules as a strategy to promote tubulin heterodimer degradationYang, Jianhong; Li, Yong; Yan, Wei; Li, Weimin; Qiu, Qiang; Ye, Haoyu; Chen, LijuanJournal of Biological Chemistry (2019), 294 (20), 8161-8170CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Clin. microtubule-targeting drugs are functionally divided into microtubule-destabilizing and microtubule-stabilizing agents. Drugs from both classes achieve microtubule inhibition by binding different sites on tubulin and inhibiting or promoting polymn. with no concomitant effects on the protein levels of tubulin heterodimers. Here, we have identified a series of small mols. with diverse structures potentially representing a third class of novel tubulin inhibitors that promote degrdn. by covalent binding to Cys-239 of β-tubulin. The small mols. highlighted in this study include T0070907 (a peroxisome proliferator-activated receptor γ inhibitor), T007-1 (a T0070907 deriv.), T138067, N,N'-ethylene-bis(iodoacetamide) (EBI), and allyl isothiocyanate (AITC). Label-free quant. proteomic anal. revealed that T007-1 promotes tubulin degrdn. with high selectivity. Mass spectrometry findings showed covalent binding of both T0070907 and T007-01 to Cys-239 of β-tubulin. Furthermore, T007-1 exerted a degradative effect on tubulin isoforms possessing Cys-239 (β2, β4, and β5(β)) but not those contg. Ser-239 (β3, β6) or mutant β-tubulin with a C239S substitution. Three small mols. (T138067, EBI, and AITC) also reported to bind covalently to Cys-239 of β-tubulin similarly induced tubulin degrdn. Our results strongly suggest that covalent modification of Cys-239 of β-tubulin by small mols. could serve as a novel strategy to promote tubulin heterodimer degrdn. We propose that these small mols. represent a third novel class of tubulin inhibitor agents that exert their effects through degrdn. activity.338Peterson, E. A.; Teffera, Y.; Albrecht, B. K.; Bauer, D.; Bellon, S. F.; Boezio, A.; Boezio, C.; Broome, M. A.; Choquette, D.; Copeland, K. W.; Dussault, I.; Lewis, R.; Lin, M. H.; Lohman, J.; Liu, J.; Potashman, M.; Rex, K.; Shimanovich, R.; Whittington, D. A.; Vaida, K. R.; Harmange, J. C. Discovery of potent and selective 8-fluorotriazolopyridine c-Met inhibitors. J. Med. Chem. 2015, 58, 2417– 2430, DOI: 10.1021/jm501913a[ACS Full Text], [CAS], Google Scholar338https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtV2nt70%253D&md5=ae86b1c6fac93caa1deecd89410bd837Discovery of Potent and Selective 8-Fluorotriazolopyridine c-Met InhibitorsPeterson, Emily A.; Teffera, Yohannes; Albrecht, Brian K.; Bauer, David; Bellon, Steven F.; Boezio, Alessandro; Boezio, Christiane; Broome, Martin A.; Choquette, Deborah; Copeland, Katrina W.; Dussault, Isabelle; Lewis, Richard; Lin, Min-Hwa Jasmine; Lohman, Julia; Liu, Jingzhou; Potashman, Michele; Rex, Karen; Shimanovich, Roman; Whittington, Douglas A.; Vaida, Karina R.; Harmange, Jean-ChristopheJournal of Medicinal Chemistry (2015), 58 (5), 2417-2430CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The overexpression of c-Met and/or hepatocyte growth factor (HGF), the amplification of the MET gene, and mutations in the c-Met kinase domain can activate signaling pathways that contribute to cancer progression by enabling tumor cell proliferation, survival, invasion, and metastasis. Herein, the authors report the discovery of 8-fluorotriazolopyridines as inhibitors of c-Met activity. Optimization of the 8-fluorotriazolopyridine scaffold through the combination of structure-based drug design, SAR studies, and metabolite identification provided potent (cellular IC50 < 10 nM) selective inhibitors of c-Met with desirable pharmacokinetic properties, e.g. I, that demonstrate potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.339Prasad, V.; Birzin, E. T.; McVaugh, C. T.; Van Rijn, R. D.; Rohrer, S. P.; Chicchi, G.; Underwood, D. J.; Thornton, E. R.; Smith, A. B., 3rd; Hirschmann, R. Effects of heterocyclic aromatic substituents on binding affinities at two distinct sites of somatostatin receptors. Correlation with the electrostatic potential of the substituents. J. Med. Chem. 2003, 46, 1858– 1869, DOI: 10.1021/jm0205088[ACS Full Text], [CAS], Google Scholar339https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXivFagt70%253D&md5=db7d7f8255fae153a3cfd9912dceb1f4Effects of Heterocyclic Aromatic Substituents on Binding Affinities at Two Distinct Sites of Somatostatin Receptors. Correlation with the Electrostatic Potential of the SubstituentsPrasad, Vidya; Birzin, Elizabeth T.; McVaugh, Cheryl T.; van Rijn, Rachel D.; Rohrer, Susan P.; Chicchi, Gary; Underwood, Dennis J.; Thornton, Edward R.; Smith, Amos B., III; Hirschmann, RalphJournal of Medicinal Chemistry (2003), 46 (10), 1858-1869CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)In our continuing program exploring glucose-based peptidomimetics of somatostatin (SRIF-14), we sought to improve the water soly. of our glycosides. This led to insights into the nature of the ligand binding sites at the SRIF receptor. Replacement of the C4 benzyl substituent in glucoside (+)-2 with pyridinylmethyl or pyrazin-2-ylmethyl congeners increased water soly. and enhanced affinity for the human SRIF subtype receptor 4 (sst4). We attribute this effect to hydrogen bond formation. The pyridin-3-ylmethyl substituent at C4, when combined with the imidazol-4-ylmethyl group at C2, generated (-)-19, which has the highest affinity of a glucose-based peptidomimetic at a human SRIF receptor to date (Ki 53 ± 23 nM, n = 6 at sst4). The C4 heterocyclic congeners of glucosides bearing a 1-methoxy substituent rather than an indole side chain at the anomeric carbon, such as (+)-16, also provided information about the Trp8 binding pocket. We correlated the SARs at both the C4 and the Trp8 binding pockets with calcns. of the electrostatic potentials of the diverse C4 arom. substituents using Spartan 3-21G(*) MO anal. These calcns. provide an approx. anal. of a mol.'s ability to interact within a receptor binding site. Our binding studies show that benzene and indole rings, but not pyridinylmethyl nor pyrazin-2-ylmethyl rings, can bind the hydrophobic Trp8 binding pocket of sst4. The Spartan 3-21G(*) MO anal. reveals significant neg. electrostatic potential in the region of the π-clouds for the benzene and indole rings but not for the pyridinylmethyl or pyrazin-2-ylmethyl congeners. Our data further demonstrate that the replacement of benzene or indole side chains by heterocyclic arom. rings typified by pyridine and pyrazine not only enhances water soly. and hydrogen bonding capacity as expected, but can also profoundly diminish the ability of the π-cloud of the arom. substituent to interact with side chains of an arom. binding pocket such as that for Trp8 of SRIF-14. Conversely, these calcns. accommodate the exptl. findings that pyrazin-2-ylmethyl and pyridinylmethyl substituents at C4- of C1-indole-substituted glycosides afford higher affinities at sst4 than the C4-benzyl group of (+)-2. This result is consistent with the high electron d. in the plane of the heterocycle depicted in Figure 6 which can accept hydrogen bonds from the C4 binding pocket of the receptor. Unexpectedly, we found that the 2-fluoropyridin-5-ylmethyl analog (+)-14 more closely resembles the binding affinity of (+)-8 than that of (+)-2, thus suggesting that (+)-14 represents a rare example of a carbon linked fluorine atom acting as a hydrogen bond acceptor. We attribute this result to the ability of the proton to bind the nitrogen and fluorine atoms simultaneously in a bifurcated arrangement. At the NK1 receptor of substance P (SP), the free hydroxyl at C4 optimizes affinity.340Leach, A. G.; Kidley, N. J. Quantitatively interpreted enhanced inhibition of cytochrome P450s by heteroaromatic rings containing nitrogen. J. Chem. Inf. Model. 2011, 51, 1048– 1063, DOI: 10.1021/ci2000506[ACS Full Text], [CAS], Google Scholar340https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXlsFyktbc%253D&md5=9b37fed557509c4c40c4521797bea92eQuantitatively Interpreted Enhanced Inhibition of Cytochrome P450s by Heteroaromatic Rings Containing NitrogenLeach, Andrew G.; Kidley, Nathan J.Journal of Chemical Information and Modeling (2011), 51 (5), 1048-1063CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)It has been known for a long time that certain substructures bind to the heme iron in cytochromes P 450. Detection of spectroscopic changes and crystal structures of protein ligand complexes have provided qual. evidence, including for arom. nitrogen-contg. ligands. Compds. contg. these same substructures are more likely to inhibit cytochrome P450s than expected due to lipophilicity. These two sets of observations are not easily linked by expt., because binding to the iron atom alone is not readily measured. Quantum mech. (d. functional) calcns. of binding energies for a no. of different arom. heterocycles to heme iron in a range of oxidn. and spin states can provide a quant. link between the obsd. structures and the biochem. inhibition that is measured. The studies reported here for a set of heteroarom. rings contg. nitrogen begin with quantum mech. calcns. which provide geometries and binding energies. Subsequently, AstraZeneca's database of cytochrome P 450 inhibition assays has been searched to find data that are relevant to the same set of heteroarom. compds. These data have been analyzed in a no. of fashions to account for both the narrow dynamic range of the assays and the lipophilicity dependence of this kind of inhibition. Finally, crystal structures have provided exptl. geometric information. Taken together these different sources suggest that binding to the metal in our inhibition assays is dominated by FeIII in its doublet state, most likely occurring when the iron is pentavalent. Computed binding energies to this state contrast with the hydrogen-bond acceptor ability and basicity of the compds., neither of which are able to correctly account for the effect of the particular environment in which the iron is found. This highlights the value of modeling biochem. events as closely as can be computationally afforded. The computational protocol devised was used to make predictions about a set of as yet unknown heteroarom. compds. suggested by Pitt et al.341Green, J.; Cao, J.; Bandarage, U. K.; Gao, H.; Court, J.; Marhefka, C.; Jacobs, M.; Taslimi, P.; Newsome, D.; Nakayama, T.; Shah, S.; Rodems, S. Design, synthesis, and structure-activity relationships of pyridine-based rho kinase (ROCK) inhibitors. J. Med. Chem. 2015, 58, 5028– 5037, DOI: 10.1021/acs.jmedchem.5b00424[ACS Full Text], [CAS], Google Scholar341https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXpsFars7k%253D&md5=7839e593cb7add8c828cbb4ac49ed674Design, Synthesis, and Structure-Activity Relationships of Pyridine-Based Rho Kinase (ROCK) InhibitorsGreen, Jeremy; Cao, Jingrong; Bandarage, Upul K.; Gao, Huai; Court, John; Marhefka, Craig; Jacobs, Marc; Taslimi, Paul; Newsome, David; Nakayama, Tomoko; Shah, Sundeep; Rodems, SteveJournal of Medicinal Chemistry (2015), 58 (12), 5028-5037CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The Rho kinases (ROCK1 and ROCK2) are highly homologous serine/threonine kinases that act on substrates assocd. with cellular motility, morphol., and contraction and are of therapeutic interest in diseases assocd. with cellular migration and contraction, such as hypertension, glaucoma, and erectile dysfunction. Beginning with compd. 4, an inhibitor of ROCK1 identified through high-throughput screening, systematic exploration of SAR, and application of structure-based design, led to potent and selective ROCK inhibitors. Compd. 37 represents significant improvements in inhibition potency, kinase selectivity, and CYP inhibition and possesses pharmacokinetics suitable for in vivo experimentation.342Dolle, F. [18F]fluoropyridines: From conventional radiotracers to the labeling of macromolecules such as proteins and oligonucleotides. Ernst Schering Res. Found. Workshop 2007, 64, 113– 157, DOI: 10.1007/978-3-540-49527-7_5343Kumar, J. S.; Walker, M.; Packiarajan, M.; Jubian, V.; Prabhakaran, J.; Chandrasena, G.; Pratap, M.; Parsey, R. V.; Mann, J. J. Radiosynthesis and in vivo evaluation of neuropeptide Y5 receptor (NPY5R) PET tracers. ACS Chem. Neurosci. 2016, 7, 540– 545, DOI: 10.1021/acschemneuro.5b00315[ACS Full Text], [CAS], Google Scholar343https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xis1Citrs%253D&md5=3b6e852390caf4c665b8a327a0045481Radiosynthesis and in Vivo Evaluation of Neuropeptide Y5 Receptor (NPY5R) PET TracersKumar, J. S. Dileep; Walker, Mary; Packiarajan, Mathivanan; Jubian, Vrej; Prabhakaran, Jaya; Chandrasena, Gamini; Pratap, Mali; Parsey, Ramin V.; Mann, J. JohnACS Chemical Neuroscience (2016), 7 (5), 540-545CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)Neuropeptide Y receptor type 5 (NPY5R) is a G-protein coupled receptor (GPCR) that belongs to the subfamily of neuropeptide receptors (NPYR) that mediate the action of endogenous neuropeptide Y (NPY). Animal models and preclin. studies indicate a role for NPY5R in the pathophysiol. of depression, anxiety, and obesity and as a target of potential therapeutic drugs. To better understand the pathophysiol. involvement of NPY5R, and to measure target occupancy by potential therapeutic drugs, it would be advantageous to measure NPY5R binding in vivo by positron emission tomog. (PET). Four potent and selective NPY5R antagonists were radiolabeled via nucleophilic arom. substitution reactions with [18F]fluoride. Of the four radioligands investigated, PET studies in anesthetized baboons showed that [18F]LuAE00654 ([18F]N-[trans-4-({[4-(2-fluoropyridin-3-yl)thiazol-2-yl]amino}methyl)cyclohexyl]propane-2-sulfonamide) penetrates blood brain barrier (BBB) and a small amt. is retained in the brain. Slow metab. of [18F]LuAE00654 was obsd. in baboon plasma. Blocking studies with a specific NPY5R antagonist demonstrated up to 60% displacement of radioactivity in striatum, the brain region with highest NPY5R binding. Our studies suggest that [18F]LuAE00654 can be a potential PET radiotracer for the quantification and occupancy studies of NPY5R drug candidates.344Fujinaga, M.; Luo, R.; Kumata, K.; Zhang, Y.; Hatori, A.; Yamasaki, T.; Xie, L.; Mori, W.; Kurihara, Y.; Ogawa, M.; Nengaki, N.; Wang, F.; Zhang, M. R. Development of a (18)F-labeled radiotracer with improved brain kinetics for positron emission tomography imaging of translocator protein (18 kDa) in ischemic brain and glioma. J. Med. Chem. 2017, 60, 4047– 4061, DOI: 10.1021/acs.jmedchem.7b00374[ACS Full Text], [CAS], Google Scholar344https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmt1amtLk%253D&md5=851513be4dbb048ec16135700e33cf36Development of a 18F-Labeled Radiotracer with Improved Brain Kinetics for Positron Emission Tomography Imaging of Translocator Protein (18 kDa) in Ischemic Brain and GliomaFujinaga, Masayuki; Luo, Rui; Kumata, Katsushi; Zhang, Yiding; Hatori, Akiko; Yamasaki, Tomoteru; Xie, Lin; Mori, Wakana; Kurihara, Yusuke; Ogawa, Masanao; Nengaki, Nobuki; Wang, Feng; Zhang, Ming-RongJournal of Medicinal Chemistry (2017), 60 (9), 4047-4061CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)We designed four novel acetamidobenzoxazolone compds. 7a-d as candidates for positron emission tomog. (PET) radiotracers for imaging the translocator protein (18 kDa, TSPO) in ischemic brain and glioma. Among these compds., 2-(5-(6-fluoropyridin-3-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamide (7d) exhibited high binding affinity (Ki = 13.4 nM) with the TSPO and moderate lipophilicity (log D = 1.92). [18F]7d was radiosynthesized by [18F]fluorination of the bromopyridine precursor 7h with [18F]F- in 12 ± 5% radiochem. yield (n = 6, decay-cor.). In vitro autoradiog. and PET studies of ischemic rat brain revealed higher binding of [18F]7d with TSPO on the ipsilateral side, as compared to the contralateral side, and improved brain kinetics compared with our previously developed radiotracers. Metabolite study of [18F]7d showed 93% of unchanged form in the ischemic brain at 30 min after injection. Moreover, PET study with [18F]7d provided a clear tumor image in a glioma-bearing rat model. We demonstrated that [18F]7d is a useful PET radiotracer for visualizing not only neuroinflammation but also glioma and will translate this radiotracer to a "first-in-human" study in our facility.345Naik, R.; Valentine, H.; Dannals, R. F.; Wong, D. F.; Horti, A. G. Synthesis and evaluation of a new (18)F-labeled radiotracer for studying the GABAB receptor in the mouse brain. ACS Chem. Neurosci. 2018, 9, 1453– 1461, DOI: 10.1021/acschemneuro.8b00038[ACS Full Text], [CAS], Google Scholar345https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjslKrtrc%253D&md5=40518bd59f1589a4346688967f279e2eSynthesis and Evaluation of a New 18F-Labeled Radiotracer for Studying the GABAB Receptor in the Mouse BrainNaik, Ravi; Valentine, Heather; Dannals, Robert F.; Wong, Dean F.; Horti, Andrew G.ACS Chemical Neuroscience (2018), 9 (6), 1453-1461CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)New GABAB agonists, fluoropyridyl ether analogs of baclofen, have been synthesized as potential PET radiotracers. The compd. with highest inhibition binding affinity as well as greatest agonist response, (R)-4-amino-3-(4-chloro-3-((2-fluoropyridin-4-yl)methoxy)phenyl)butanoic acid (1b), was radiolabeled with 18F with good radiochem. yield, high radiochem. purity, and high molar radioactivity. The regional brain distribution of the radiolabeled (R)-4-amino-3-(4-chloro-3-((2-[18F]fluoropyridin-4-yl)methoxy)phenyl)butanoic acid, [18F]1b, was studied in CD-1 male mice. The study demonstrated that [18F]1b enters the mouse brain (1% ID/g tissue). The accumulation of [18F]1b in the mouse brain was inhibited (35%) by preinjection of GABAB agonist 1a, suggesting that the radiotracer brain uptake is partially mediated by GABAB receptors. The presented data demonstrate a feasibility of imaging of GABAB receptors in rodents and justify further development of GABAB PET tracers with improved specific binding and greater blood-brain barrier permeability.346Ward, S. E.; Harries, M.; Aldegheri, L.; Andreotti, D.; Ballantine, S.; Bax, B. D.; Harris, A. J.; Harker, A. J.; Lund, J.; Melarange, R.; Mingardi, A.; Mookherjee, C.; Mosley, J.; Neve, M.; Oliosi, B.; Profeta, R.; Smith, K. J.; Smith, P. W.; Spada, S.; Thewlis, K. M.; Yusaf, S. P. Discovery of N-[(2S)-5-(6-fluoro-3-pyridinyl)-2,3-dihydro-1H-inden-2-yl]-2-propanesulfonamide, a novel clinical AMPA receptor positive modulator. J. Med. Chem. 2010, 53, 5801– 5812, DOI: 10.1021/jm1005429[ACS Full Text], [CAS], Google Scholar346https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXos1ektbk%253D&md5=b10f279d94da22aa0662d4b8a9999b5eDiscovery of N-[(2S)-5-(6-Fluoro-3-pyridinyl)-2,3-dihydro-1H-inden-2-yl]-2-propanesulfonamide, a Novel Clinical AMPA Receptor Positive ModulatorWard, Simon E.; Harries, Mark; Aldegheri, Laura; Andreotti, Daniele; Ballantine, Stuart; Bax, Benjamin D.; Harris, Andrew J.; Harker, Andy J.; Lund, Jesper; Melarange, Rosemary; Mingardi, Anna; Mookherjee, Claudette; Mosley, Julie; Neve, Marta; Oliosi, Beatrice; Profeta, Roberto; Smith, Kathrine J.; Smith, Paul W.; Spada, Simone; Thewlis, Kevin M.; Yusaf, Shahnaz P.Journal of Medicinal Chemistry (2010), 53 (15), 5801-5812CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of AMPA receptor pos. allosteric modulators has been optimized from poorly penetrant leads to identify mols. with excellent preclin. pharmacokinetics and CNS penetration. These discoveries led to 17i (I), a potent, efficacious CNS penetrant mol. with an excellent pharmacokinetic profile across preclin. species, which is well tolerated and is also orally bioavailable in humans.347Li, P.; Zheng, H.; Zhao, J.; Zhang, L.; Yao, W.; Zhu, H.; Beard, J. D.; Ida, K.; Lane, W.; Snell, G.; Sogabe, S.; Heyser, C. J.; Snyder, G. L.; Hendrick, J. P.; Vanover, K. E.; Davis, R. E.; Wennogle, L. P. Discovery of potent and selective inhibitors of phosphodiesterase 1 for the treatment of cognitive impairment associated with neurodegenerative and neuropsychiatric diseases. J. Med. Chem. 2016, 59, 1149– 1164, DOI: 10.1021/acs.jmedchem.5b01751[ACS Full Text], [CAS], Google Scholar347https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1aisLk%253D&md5=73741521722dc906869ffa3504ceaf2eDiscovery of Potent and Selective Inhibitors of Phosphodiesterase 1 for the Treatment of Cognitive Impairment Associated with Neurodegenerative and Neuropsychiatric DiseasesLi, Peng; Zheng, Hailin; Zhao, Jun; Zhang, Lei; Yao, Wei; Zhu, Hongwen; Beard, J. David; Ida, Koh; Lane, Weston; Snell, Gyorgy; Sogabe, Satoshi; Heyser, Charles J.; Snyder, Gretchen L.; Hendrick, Joseph P.; Vanover, Kimberly E.; Davis, Robert E.; Wennogle, Lawrence P.Journal of Medicinal Chemistry (2016), 59 (3), 1149-1164CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A diverse set of 3-aminopyrazolo[3,4-d]pyrimidinones was designed and synthesized. The structure-activity relationships of these polycyclic compds. as phosphodiesterase 1 (PDE1) inhibitors were studied along with their physicochem. and pharmacokinetic properties. Systematic optimizations of this novel scaffold culminated in the identification of a clin. candidate I, which exhibited picomolar inhibitory potency for PDE1, demonstrated excellent selectivity against all other PDE families and showed good efficacy in vivo. Currently, this investigational new drug is in Phase I clin. development and being considered for the treatment of several indications including cognitive deficits assocd. with schizophrenia and Alzheimer's disease, movement disorders, attention deficit and hyperactivity disorders, and other central nervous system (CNS) and non-CNS disorders.348Wittman, M. D.; Carboni, J. M.; Yang, Z.; Lee, F. Y.; Antman, M.; Attar, R.; Balimane, P.; Chang, C.; Chen, C.; Discenza, L.; Frennesson, D.; Gottardis, M. M.; Greer, A.; Hurlburt, W.; Johnson, W.; Langley, D. R.; Li, A.; Li, J.; Liu, P.; Mastalerz, H.; Mathur, A.; Menard, K.; Patel, K.; Sack, J.; Sang, X.; Saulnier, M.; Smith, D.; Stefanski, K.; Trainor, G.; Velaparthi, U.; Zhang, G.; Zimmermann, K.; Vyas, D. M. Discovery of a 2,4-disubstituted pyrrolo[1,2-f][1,2,4]triazine inhibitor (BMS-754807) of insulin-like growth factor receptor (IGF-1R) kinase in clinical development. J. Med. Chem. 2009, 52, 7360– 7363, DOI: 10.1021/jm900786r[ACS Full Text], [CAS], Google Scholar348https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtFOiurjJ&md5=be0fe9adea18595b4ba6f232726ba4dfDiscovery of a 2,4-Disubstituted Pyrrolo[1,2-f][1,2,4]triazine Inhibitor (BMS-754807) of Insulin-like Growth Factor Receptor (IGF-1R) Kinase in Clinical DevelopmentWittman, Mark D.; Carboni, Joan M.; Yang, Zheng; Lee, Francis Y.; Antman, Melissa; Attar, Ricardo; Balimane, Praveen; Chang, Chiehying; Chen, Cliff; Discenza, Lorell; Frennesson, David; Gottardis, Marco M.; Greer, Ann; Hurlburt, Warren; Johnson, Walter; Langley, David R.; Li, Aixin; Li, Jianqing; Liu, Peiying; Mastalerz, Harold; Mathur, Arvind; Menard, Krista; Patel, Karishma; Sack, John; Sang, Xiaopeng; Saulnier, Mark; Smith, Daniel; Stefanski, Kevin; Trainor, George; Velaparthi, Upender; Zhang, Guifen; Zimmermann, Kurt; Vyas, Dolatrai M.Journal of Medicinal Chemistry (2009), 52 (23), 7360-7363CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)This report describes the biol. activity, characterization, and SAR leading to 9d (BMS-754807, I) a small mol. IGF-1R kinase inhibitor in clin. development.349Ndubaku, C. O.; Crawford, T. D.; Chen, H.; Boggs, J. W.; Drobnick, J.; Harris, S. F.; Jesudason, R.; McNamara, E.; Nonomiya, J.; Sambrone, A.; Schmidt, S.; Smyczek, T.; Vitorino, P.; Wang, L.; Wu, P.; Yeung, S.; Chen, J.; Chen, K.; Ding, C. Z.; Wang, T.; Xu, Z.; Gould, S. E.; Murray, L. J.; Ye, W. Structure-based design of GNE-495, a potent and selective MAP4K4 inhibitor with efficacy in retinal angiogenesis. ACS Med. Chem. 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(American Chemical Society)Diverse biol. roles for mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) have necessitated the identification of potent inhibitors to study its function in various disease contexts. In particular, compds. that can be used to carry out such studies in vivo would be crit. for elucidating the potential for therapeutic intervention. A structure-based design effort coupled with property-guided optimization directed at minimizing the ability of the inhibitors to cross into the CNS led to an advanced compd. (I; GNE-495) that showed excellent potency and good PK and was used to demonstrate in vivo efficacy in a retinal angiogenesis model recapitulating effects that were obsd. in the inducible Map4k4 knockout mice.350Clark, H. R.; Beth, L. D.; Burton, R. M.; Garrett, D. L.; Miller, A. L.; Muscio, O. J., Jr. Kinetic study of the acid-promoted hydrolysis of some representative 2-fluoro nitrogen heterocycles. J. Org. Chem. 1981, 46, 4363– 4369, DOI: 10.1021/jo00335a008[ACS Full Text], [CAS], Google Scholar350https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXls1yhu70%253D&md5=e57054f89598411d7cb8064e789bc9bcKinetic study of the acid-promoted hydrolysis of some representative 2-fluoronitrogen heterocyclesClark, H. R.; Beth, L. D.; Burton, R. M.; Garrett, D. L.; Miller, A. L.; Muscio, O. J., Jr.Journal of Organic Chemistry (1981), 46 (22), 4363-9CODEN: JOCEAH; ISSN:0022-3263.The acid-promoted hydrolyses of the 2-fluoro derivs. of pyridine, the four isomeric picolines, quinoline, pyrimidine, 4-methylpyrimidine, and 4,6-dimethylpyrimidine were studied in 0.05-8.0 F HCl. At each acid concn., the reactions followed pseudo-1st-order kinetics, and at low concns. of acid, the rate of reaction increased linearly with h0. However, at higher acid concns. neg. deviations from linearity were obsd. for all the substrates and rate max. for all but the pyrimidines. These results were correlated with the decline in water activity by means of the Bunnett w and w* relationships, as well as the Bunnett-Olsen LFER. The slopes of these correlations were suggestive of a proton-transfer role for water in the reactions of the less activated 2-fluoropyridines and of 2-fluoroquinoline, while the correlations indicate a nucleophilic role for water in the reactions of the more highly activated pyrimidines. Entropies of activation were significantly more neg. for the pyridine and quinoline systems than for the pyrimidines. The results are interpreted as consistent with nucleophilic attack by water in the rate-detg. step for the reaction of the pyrimidines, while for the less activated substrates nucleophilic attack may be assisted by proton transfer to addnl. water mols.351Inoue, K.; Ohe, T.; Mori, K.; Sagara, T.; Ishii, Y.; Chiba, M. Aromatic substitution reaction of 2-chloropyridines catalyzed by microsomal glutathione S-transferase 1. Drug Metab. Dispos. 2009, 37, 1797– 1800, DOI: 10.1124/dmd.109.027698[Crossref], [PubMed], [CAS], Google Scholar351https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVOrsrnN&md5=080edfa7114d4b506d3376e514b11ab7Aromatic substitution reaction of 2-chloropyridines catalyzed by microsomal glutathione S-transferase 1Inoue, Kazuko; Ohe, Tomoyuki; Mori, Kenichi; Sagara, Takeshi; Ishii, Yasuyuki; Chiba, MasatoDrug Metabolism and Disposition (2009), 37 (9), 1797-1800CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)We investigated the substitution reaction of a series of 2-chloropyridine derivs. catalyzed by rat liver microsomal glutathione S-transferase 1. Various 2-chloropyridine derivs. were metabolized to the corresponding substituted glutathione conjugates via displacement of chlorine atom with glutathione. The reaction was affected by the electron-withdrawing strength and position of the substituents. MO calcns. on the change in Gibbs free energy between the initial and transition states verified the presence of a Meisenheimer complex and its influence on the reaction rate.352Earl, R. A.; Myers, M. J.; Johnson, A. L.; Scribner, R. M.; Wuonola, M. A.; Boswell, G. A.; Wilkerson, W. W.; Nickolson, V. J.; Tam, S. W.; Brittelli, D. R.; Chorvat, R. J.; Zaczek, R.; Cook, L.; Wang, C.; Zhang, X.; Lan, R.; Mi, B.; Wenting, H. Acetylcholine-releasing agents as cognition enhancers. Structure-activity relationships of pyridinyl pendant groups on selected core structures. Bioorg. Med. Chem. Lett. 1992, 2, 851– 854, DOI: 10.1016/S0960-894X(00)80543-0[Crossref], [CAS], Google Scholar352https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXkvVGns74%253D&md5=1d38c0fff1828d31e48c4f84d19fe783Acetylcholine-releasing agents as cognition enhancers. Structure-activity relationships of pyridinyl pendant groups on selected core structuresEarl, Richard A.; Myers, Melvyn J.; Johnson, Alexander L.; Scribner, Richard M.; Wuonola, Mark A.; Boswell, George A.; Wilkerson, Wendell W.; Nickolson, Victor J.; Tam, S. William; et al.Bioorganic & Medicinal Chemistry Letters (1992), 2 (8), 851-4CODEN: BMCLE8; ISSN:0960-894X.A no. of analogs of the cognition enhancing agent DuP 996 (I) were prepd. by varying the core structure and pendant groups in an independent fashion. The structure-activity relations of 2-, 3-, and 4-pyridinylmethyl groups as pendant groups on selected cores was examd.353Wilkerson, W. W.; Kergaye, A. A.; Tam, S. W. 3-Substituted, 3-(4-pyridinylmethyl)-1,3-dihydro-1-phenyl-2H-indol-2-ones as acetylcholine release enhancers: synthesis and SAR. J. Med. Chem. 1993, 36, 2899– 2907, DOI: 10.1021/jm00072a009[ACS Full Text], [CAS], Google Scholar353https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXjtVKmuw%253D%253D&md5=f947153ab7977c773445f31b3e559ee13-Substituted 3-(4-pyridinylmethyl)-1,3-dihydro-1-phenyl-2H-indol-2-ones as acetylcholine release enhancers: synthesis and SARWilkerson, Wendell W.; Kergaye, Ahmed A.; Tam, S. WilliamJournal of Medicinal Chemistry (1993), 36 (20), 2899-907CODEN: JMCMAR; ISSN:0022-2623.A series of 3-substituted 3-(4-pyridinylmethyl)-1,3-dihydro-1-phenyl-2H-indol-2-ones I [R = H, CH2CO2Et, OH, (CH2)3CONH2, CH2CH:CHCO2Et, (CH2)5Me, etc.] was synthesized and found to enhance the stimulus-induced release of neurotransmitter acetylcholine (AcCh), and by doing so, might be useful in treating cognitive disorders where the level of this neurotransmitter may be diminished in the brain, as in Alzheimer's disease. An attempt has been made to correlate the structure of the 3-substitution with the ability of the compds. to enhance the release of AcCh from the striatum region of rat brain prepns. Alkylation of I (R = H) or its hydrochloride salt gave several of the compds.354Earl, R. A.; Zaczek, R.; Teleha, C. A.; Fisher, B. N.; Maciag, C. M.; Marynowski, M. E.; Logue, A. R.; Tam, S. W.; Tinker, W. J.; Huang, S. M.; Chorvat, R. J. 2-Fluoro-4-pyridinylmethyl analogues of linopirdine as orally active acetylcholine release-enhancing agents with good efficacy and duration of action. J. Med. Chem. 1998, 41, 4615– 4622, DOI: 10.1021/jm9803424[ACS Full Text], [CAS], Google Scholar354https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXmsVKqu7w%253D&md5=f5af93f21d02feedf99d077b178e76fb2-Fluoro-4-pyridinylmethyl Analogs of Linopirdine as Orally Active Acetylcholine Release-Enhancing Agents with Good Efficacy and Duration of ActionEarl, Richard A.; Zaczek, Robert; Teleha, Christopher A.; Fisher, Barbara N.; Maciag, Carla M.; Marynowski, Maria E.; Logue, Andrew R.; Tam, S. William; Tinker, William J.; Huang, Shiew-Mei; Chorvat, Robert J.Journal of Medicinal Chemistry (1998), 41 (23), 4615-4622CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)In an effort to improve the pharmacokinetic and pharmacodynamic properties of the cognition-enhancer linopirdine (DuP 996), a no. of core structure analogs were prepd. in which the 4-pyridyl pendant group was systematically replaced with 2-fluoro-4-pyridyl. This strategy resulted in the discovery of several compds. with improved activity in acetylcholine (ACh) release-enhancing assays, in vitro and in vivo. The most effective compd. resulting from these studies, 10,10-bis[(2-fluoro-4-pyridinyl)methyl]-9(10H)-anthracenone (I), is between 10 and 20 times more potent than linopirdine in increasing extracellular hippocampal ACh levels in the rat with a min. ED of 1 mg/kg. In addn. to superior potency, I possesses an improved pharmacokinetic profile compared to that of linopirdine. The half-life of I (2 h) in rats is 4-fold greater than that of linopirdine (0.5 h), and it showed a 6-fold improvement in brain-plasma distribution over linopirdine. On the basis of its pharmacol., pharmacokinetic, absorption, and distribution properties, I (DMP543) has been advanced for clin. evaluation as a potential palliative therapeutic for treatment of Alzheimer's disease.355Shaw, E.; Bernstein, J.; Losee, K.; Lott, W. A. Analogs of aspergillic acid. IV. Substituted 2-bromopyridine-N-oxides and their conversion to cyclic thiohydroxamic acids. J. Am. Chem. Soc. 1950, 72, 4362– 4364, DOI: 10.1021/ja01166a008[ACS Full Text], [CAS], Google Scholar355https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaG3MXis12qug%253D%253D&md5=f4f6deb3ae835708ce3a62213568875dAnalogs of aspergillic acid. IV. Substituted 2-bromopyridine N-oxides and their conversion to cyclic thiohydroxamic acidsShaw, Elliott; Bernstein, Jack; Losee, Kathryn; Lott, W. A.Journal of the American Chemical Society (1950), 72 (), 4362-4CODEN: JACSAT; ISSN:0002-7863.cf. C.A. 43, 8382g. Reaction of 1.0 mole 2-bromopyridine (I) and 1.2 moles BzO2H in CHCl3 4 days at room temp. and concn. of the 20% aq. HCl ext. gave 60% 2-bromopyridine 1-oxide-HCl (II), m. 135-6°. Derivs. of II prepd. similarly (read yield (%) and m.p.): 3-Me, 67, 179-80°; 4-Me, 59, 147-8°; 5-Me, 64, 141-2°; 6-Me, 61, 185-6°; 3-EtO, 52, 159-60°; 5-Br, 6.5, 165-6°. 2-Bromo-5-methylpyridine, prepd. in 77% yield, b14 97°, m. 42-3°; 6-Me isomer, b9 77°. Slow addn. of 1.5 moles 40% AcO2H to 1 mole I at 10-40°, heating 24 hrs. at 45-50°, concn. at 30° to 0.5 vol., addn. to ice and excess 40% KOH at 5°, CHCl3 extn., etc., gave 70% II. Neutralization of 0.31 mole II in 75 cc. H2O with 25% NaOH, portionwise addn. of 32 g. NaHSO3 in 150 cc. H2O at 100° during 1 hr., heating an addnl. 30 min. at 100°, cooling, and acidification of the filtrate with 6 N HCl yielded 61% 1-hydroxy-2(1H)-pyridinethione (III), m. 68-70°, gives a deep blue with FeCl3. III was also obtained from equimol. amts. of II and Na2S. The minimal inhibiting concns. of III in γ/cc. against Staphylococcus aureus P209, Klebsiella pneumoniae, and the bacillus of Calmette and Guerin were 0.06, 1.5, and 0.006, resp. Similar data for aspergillic acid were 20, 30, and 4 and for 1-hydroxy-2(1H)-pyridone (IV), 3, 40, and 2. Derivs. of III (read yield (%), m.p., and min. inhibiting concn.): 3-Me, 52, 74-5°, 0.06, 0.6, 0.004; 4-Me, 60, 59-61°, 0.08, 1.5, 0.001; 5-Me, 53, 106-7°, 0.07, 1.5, 0.001; 6-Me, 50%, 52-4°, 0.1, 3.5, 0.003; 3-EtO, 85, 101-3°, 0.08, 1.5, 0.03; 5-Br, 40%, 130-1°, 0.1, 2.0, 0.008. Refluxing II and 1 mole thiourea in abs. EtOH 1 hr. gave 72% 2-pyridyl-N-oxide-isothiourea HBr salt [2-(guanylmercapto)pyridine 1-oxide-HBr], m. 160-0.5° (decompn.), converted by 8% Na2CO3 in 4 hrs. at room temp. to 78% III. Heating III in 10% NaOH at 100° 1.5 hrs., concn. of the acidified soln. in vacuo, soln. of the residue in H2O, and addn. of aq. Cu(OAc)2 gave 53% Cu salt of IV. Warming 0.03 mole PhCH2SH, 0.012 mole II, and 0.043 Na in abs. EtOH 1 hr. at 50° letting stand 2 hrs. at room temp., addn. of excess NaOH, and EtOAc extn. gave 40% 2-(benzylmercapto)pyridine 1-oxide, m. 167-9°. BzO2H reacted quantitatively with 1 mole 2-benzylmercaptopyridine in CHCl3 to give 68% 2-pyridyl benzyl sulfoxide, m. 87-8°.356Sarantakis, D.; Sutherland, J. K.; Tortorella, C.; Tortorella, V. 2-Fluoropyridine N-oxide and its reactions with amino-acid derivatives. J. Chem. Soc. C 1968, 1, 72– 73, DOI: 10.1039/j39680000072357Chen, J.-G.; Markovitz, D. A.; Yang, A. Y.; Rabel, S. R.; Pang, J.; Dolinsky, O.; Wu, L.-S.; Alasandro, M. Degradation of a fluoropyridinyl drug in capsule formulation: degradant identification, proposed degradation mechanism, and formulation optimization. Pharm. Dev. Technol. 2000, 5, 561– 570, DOI: 10.1081/PDT-100102039[Crossref], [PubMed], [CAS], Google Scholar357https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXosV2nurc%253D&md5=aa625c3196719513f726a3dd5e2676a2Degradation of a fluoropyridinyl drug in capsule formulation: degradant identification, proposed degradation mechanism, and formulation optimizationChen, Jian-Ge; Markovitz, Debbie A.; Yang, Amy Y.; Rabel, Shelley R.; Pang, Josephine; Dolinsky, Olga; Wu, Lei-Shu; Alasandro, MarkPharmaceutical Development and Technology (2000), 5 (4), 561-570CODEN: PDTEFS; ISSN:1083-7450. (Marcel Dekker, Inc.)The purpose of this paper was to investigate the degrdn. chem. of a fluoropyridinyl drug candidate in capsule formulation and to optimize the formulation based on a proposed degrdn. mechanism. Small developmental batches of capsules were made by trituration of the drug and excipients by using a mortar and pestle, followed by manual encapsulation. Degradants were identified by LC-MS/MS and LC-photodiode array detector (PDA) and were monitored by LC-UV detector (UVD) during stability studies. The drug could undergo a nucleophilic substitution reaction in which hydroxyl groups replace the fluorine substituents on the pyridine rings. The initial degrdn. rate is independent of the drug concn. but dependent on the temp., the pH of the microenvironment, and the excipient type. On the basis of these exptl. results, a nucleophilic substitution reaction mechanism for the degrdn. was proposed and a successful capsule formulation was developed.358Rabel, S. R.; Shinwari, M. K.; Nemeth, G. A.; Blom, K. F.; Maurin, M. B. Characterization of the solution stability and degradation products of the nover neurotransmitter release enhancer 10,10-bis(2-fluoro-4-pyridinylmethyl)-9(10H)-anthracenone. Drug Stability 1997, 1, 224– 230[CAS], Google Scholar358https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXnvFKqtL0%253D&md5=46005c211cec07c5c63047a040a3e589Characterization of the solution stability and degradation products of the novel neurotransmitter release enhancer 10,10-bis(2-fluoro-4-pyridinylmethyl)-9(10H)-anthracenone (DMP 543)Rabel, Shelley R.; Shinwari, Mirwais K.; Nemeth, Gregory A.; Blom, Karl F.; Maurin, Michael B.Drug Stability (1997), 1 (4), 224-230CODEN: DRSTFY; ISSN:1355-5618. (Radcliffe Medical Press)The soln. stability of a neurotransmitter release enhancer, (10,10-bis(2-fluoro-4-pyridinylmethyl)-9(10H)-anthracenone) (DMP 543), was examd. as a function of pH and buffer concn. at 80°, μ = 0.5. DMP 543 underwent general and specific acid catalysis below pH 7, resulting in 2 major degrdn. products, while the compd. was stable at pH 7-9 after 51 days at 80°. The degradants were isolated by semi-preparative HPLC and characterized by proton, carbon and fluorine NMR and mass spectrometry. The major route of degrdn. was the sequential hydrolysis of the pyridine rings on the mol. to form the 2-hydroxy substituted products.359Snyder, G. L.; Prickaerts, J.; Wadenberg, M. L.; Zhang, L.; Zheng, H.; Yao, W.; Akkerman, S.; Zhu, H.; Hendrick, J. P.; Vanover, K. E.; Davis, R.; Li, P.; Mates, S.; Wennogle, L. P. Preclinical profile of ITI-214, an inhibitor of phosphodiesterase 1, for enhancement of memory performance in rats. Psychopharmacology (Berl.) 2016, 233, 3113– 3124, DOI: 10.1007/s00213-016-4346-2[Crossref], [PubMed], [CAS], Google Scholar359https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2s7jslaksA%253D%253D&md5=6fda37a7d71613a653ddebb26d8df313Preclinical profile of ITI-214, an inhibitor of phosphodiesterase 1, for enhancement of memory performance in ratsSnyder Gretchen L; Zhang Lei; Zheng Hailin; Yao Wei; Hendrick Joseph P; Vanover Kimberly E; Davis Robert; Li Peng; Mates Sharon; Wennogle Lawrence P; Prickaerts Jos; Akkerman Sven; Wadenberg Marie-Louise; Zhu HongwenPsychopharmacology (2016), 233 (17), 3113-24 ISSN:.RATIONALE: Therapeutic agents for memory enhancement in psychiatric disorders, such as schizophrenia, are urgently needed. OBJECTIVE: The aim of this study is to characterize the preclinical profile of ITI-214, a potent inhibitor of phosphodiesterase 1 (PDE1). METHODS: ITI-214 was assayed for inhibition of PDE1 versus other PDE enzyme families using recombinant human PDE enzymes and for off-target binding to 70 substrates (General SEP II diversity panel; Caliper Life Sciences). Effects of ITI-214 (0.1-10 mg/kg, po) on memory performance were assayed in rats using the novel object recognition (NOR) paradigm, with drug given at specified time points prior to or following exposure to objects in an open field. ITI-214 was evaluated for potential drug-drug interaction with risperidone in rats using conditioned avoidance response (CAR) and pharmacokinetic assessments. RESULTS: ITI-214 inhibited PDE1A (K i = 33 pmol) with >1000-fold selectivity for the nearest other PDE family (PDE4D) and displayed minimal off-target binding interactions in a 70-substrate selectivity profile. By using specific timing of oral ITI-214 administration, it was demonstrated in the NOR that ITI-214 is able to enhance acquisition, consolidation, and retrieval memory processes. All memory effects were in the absence of effects on exploratory behavior. ITI-214 did not disrupt the risperidone pharmacokinetic profile or effects in CAR. CONCLUSIONS: ITI-214 improved the memory processes of acquisition, consolidation, and retrieval across a broad dose range (0.1-10 mg/kg, po) without disrupting the antipsychotic-like activity of a clinical antipsychotic medication, specifically risperidone. Clinical development of ITI-214 is currently in progress.360Lee, Y. S.; Chuang, S. H.; Huang, L. Y.; Lai, C. L.; Lin, Y. H.; Yang, J. Y.; Liu, C. W.; Yang, S. C.; Lin, H. S.; Chang, C. C.; Lai, J. Y.; Jian, P. S.; Lam, K.; Chang, J. M.; Lau, J. Y.; Huang, J. J. Discovery of 4-aryl-N-arylcarbonyl-2-aminothiazoles as Hec1/Nek2 inhibitors. Part I: optimization of in vitro potencies and pharmacokinetic properties. J. Med. Chem. 2014, 57, 4098– 4110, DOI: 10.1021/jm401990s[ACS Full Text], [CAS], Google Scholar360https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXntFChtLw%253D&md5=e583b657fa4837f99545e596f08b6168Discovery of 4-Aryl-N-arylcarbonyl-2-aminothiazoles as Hec1/Nek2 Inhibitors. Part I: Optimization of in Vitro Potencies and Pharmacokinetic PropertiesLee, Ying-Shuan E.; Chuang, Shih-Hsien; Huang, Lynn Y. L.; Lai, Chun-Liang; Lin, Yu-Hsiang; Yang, Ju-Ying; Liu, Chia-Wei; Yang, Sheng-chuan; Lin, Her-Sheng; Chang, Chia-chi; Lai, Jun-Yu; Jian, Pei-Shiou; Lam, King; Chang, Jia-Ming; Lau, Johnson Y. N.; Huang, Jiann-JyhJournal of Medicinal Chemistry (2014), 57 (10), 4098-4110CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of 4-aryl-N-arylcarbonyl-2-aminothiazoles was designed and synthesized as Hec1/Nek2 inhibitors. Structural optimization led to compd. I bearing C-4' 4-methoxyphenoxy and 4-(o-fluoropyridyl)carbonyl groups that showed low nanomolar in vitro antiproliferative activity (IC50: 16.3-42.7 nM), high i.v. AUC (64.9 μM·h, 2.0 mg/kg) in SD rats, and significant in vivo antitumor activity (T/C = 32%, 20 mg/kg, IV) in mice bearing human MDA-MB-231 xenografts. Cell responses resulting from Hec1/Nek2 inhibition were obsd. in cells treated with I, including a reduced level of Hec1 coimmunopptd. with Nek2, degrdn. of Nek2, mitotic abnormalities, and apoptosis. Compd. I showed selectivity toward cancer cells over normal phenotype cells and was inactive in a [3H]astemizole competitive binding assay for hERG liability screening. Therefore, I is as a good lead toward the discovery of a preclin. candidate targeting Hec1/Nek2 interaction.361Johnson, C. M.; Linsky, T. W.; Yoon, D. W.; Person, M. D.; Fast, W. Discovery of halopyridines as quiescent affinity labels: inactivation of dimethylarginine dimethylaminohydrolase. J. Am. Chem. Soc. 2011, 133, 1553– 1562, DOI: 10.1021/ja109207m[ACS Full Text], [CAS], Google Scholar361https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjvFGgtA%253D%253D&md5=edfd01568d3bc3dca9d3f017cdc27cc4Discovery of Halopyridines as Quiescent Affinity Labels: Inactivation of Dimethylarginine DimethylaminohydrolaseJohnson, Corey M.; Linsky, Thomas W.; Yoon, Dae-Wi; Person, Maria D.; Fast, WalterJournal of the American Chemical Society (2011), 133 (5), 1553-1562CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)In an effort to develop novel covalent modifiers of dimethylarginine dimethylaminohydrolase (DDAH) that are useful for biol. applications, a set of "fragment"-sized inhibitors that were identified using a high-throughput screen are tested for time-dependent inhibition. One structural class of inactivators, 4-halopyridines, show time- and concn.-dependent inactivation of DDAH, and the inactivation mechanism of one example, 4-bromo-2-methylpyridine (1), is characterized in detail. The neutral form of halopyridines is not very reactive with excess glutathione. However, 1 readily reacts, with loss of its halide, in a selective, covalent, and irreversible manner with the active-site Cys249 of DDAH. This active-site Cys is not particularly reactive (pKa ca. 8.8), and 1 does not inactivate papain (Cys pKa ca. ≤4), suggesting that, unlike many reagents, Cys nucleophilicity is not a predominating factor in selectivity. Rather, binding and stabilization of the more reactive pyridinium form of the inactivator by a second moiety, Asp66, is required for facile reaction. This constraint imparts a unique selectivity profile to these inactivators. To our knowledge, halopyridines have not previously been reported as protein modifiers, and therefore they represent a first-in-class example of a novel type of quiescent affinity label.362Johnson, C. M.; Monzingo, A. F.; Ke, Z.; Yoon, D. W.; Linsky, T. W.; Guo, H.; Robertus, J. D.; Fast, W. On the mechanism of dimethylarginine dimethylaminohydrolase inactivation by 4-halopyridines. J. Am. Chem. Soc. 2011, 133, 10951– 10959, DOI: 10.1021/ja2033684[ACS Full Text], [CAS], Google Scholar362https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnvFagsrg%253D&md5=1c51b818e7ab51bb57831412e317c533On the Mechanism of Dimethylarginine Dimethylaminohydrolase Inactivation by 4-HalopyridinesJohnson, Corey M.; Monzingo, Arthur F.; Ke, Zhihong; Yoon, Dae-Wi; Linsky, Thomas W.; Guo, Hua; Robertus, Jon D.; Fast, WalterJournal of the American Chemical Society (2011), 133 (28), 10951-10959CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Small mols. capable of selective covalent protein modification are of significant interest for the development of biol. probes and therapeutics. We recently reported that 2-methyl-4-bromopyridine is a quiescent affinity label for the nitric oxide controlling enzyme dimethylarginine dimethylaminohydrolase (DDAH). Discovery of this novel protein modifier raised the possibility that the 4-halopyridine motif may be suitable for wider application. Therefore, the inactivation mechanism of the related compd. 2-hydroxymethyl-4-chloropyridine is probed here in more detail. Soln. studies support an inactivation mechanism in which the active site Asp66 residue stabilizes the pyridinium form of the inactivator, which has enhanced reactivity toward the active site Cys, resulting in covalent bond formation, loss of the halide, and irreversible inactivation. A 2.18 Å resoln. X-ray crystal structure of the inactivated complex elucidates the orientation of the inactivator and its covalent attachment to the active site Cys, but the structural model does not show an interaction between the inactivator and Asp66. Mol. modeling is used to investigate inactivator binding, reaction, and also a final pyridinium deprotonation step that accounts for the apparent differences between the soln.-based and structural studies with respect to the role of Asp66. This work integrates multiple approaches to elucidate the inactivation mechanism of a novel 4-halopyridine "warhead," emphasizing the strategy of using pyridinium formation as a "switch" to enhance reactivity when bound to the target protein.363Madhura, D. B.; Liu, J.; Meibohm, B.; Lee, R. E. Phase II metabolic pathways of spectinamide antitubercular agents: a comparative study of the reactivity of 4-substituted pyridines to glutathione conjugation. MedChemComm 2016, 7, 114– 117, DOI: 10.1039/C5MD00349K[Crossref], [PubMed], [CAS], Google Scholar363https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVGisrrE&md5=1c280109f629d3a6c6300d036fabc874Phase II metabolic pathways of spectinamide antitubercular agents: a comparative study of the reactivity of 4-substituted pyridines to glutathione conjugationMadhura, Dora B.; Liu, Jiuyu; Meibohm, Bernd; Lee, Richard E.MedChemComm (2016), 7 (1), 114-117CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)Spectinamides are promising new semisynthetic anti-tubercular agents that are modified with a pyridyl side chain, which blocks native efflux from the tuberculosis cell. This study, describes the stability of an advanced panel of spectinamide analogs, with varying substitutions to the pyridyl side chain, to Phase-II conjugative metab. by glucuronosyl transferase, sulfotransferase and glutathione-S-transferase enzymes using both human and rat S9 enzyme fractions. All solely 5-substituted pyridyl spectinamides exhibited complete stability towards Phase II conjugative enzymes. However, 4-chloro substituted pyridyl spectinamides were susceptible to glutathione conjugation with rates dependent on other substitutions to the pyridine ring. Electron donating 5-substitutions increased the propensity for glutathione conjugation and conversely the introduction of an electron withdrawing 5-fluoro group blocked all obsd. glutathione conjugation. Based on these Phase II metab. studies, lead spectinamides 1329, 1445, 1599, 1661 and 1810 were found to have favorable properties for potential lead compds. with no Phase II liabilities.364Okamura, T.; Kikuchi, T.; Okada, M.; Toramatsu, C.; Fukushi, K.; Takei, M.; Irie, T. Noninvasive and quantitative assessment of the function of multidrug resistance-associated protein 1 in the living brain. J. Cereb. Blood Flow Metab. 2009, 29, 504– 511, DOI: 10.1038/jcbfm.2008.135[Crossref], [PubMed], [CAS], Google Scholar364https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXisVSnsr8%253D&md5=0567e4c206f6481c8375f4e016f0f2b5Noninvasive and quantitative assessment of the function of multidrug resistance-associated protein 1 in the living brainOkamura, Toshimitsu; Kikuchi, Tatsuya; Okada, Maki; Toramatsu, Chie; Fukushi, Kiyoshi; Takei, Makoto; Irie, ToshiakiJournal of Cerebral Blood Flow & Metabolism (2009), 29 (3), 504-511CODEN: JCBMDN; ISSN:0271-678X. (Nature Publishing Group)Multidrug resistance-assocd. protein 1 (MRP1) acts as a defense mechanism by pumping xenobiotics and endogenous metabolites out of the brain. The currently available techniques for studying brain-to-blood efflux have significant limitations related to either their invasiveness or the qual. assessment. Here, we describe an in vivo method, which overcomes these limitations for assessing MRP1 function, using positron emission tomog. (PET) and a PET probe. 6-Bromo-7-[11C]methylpurine was designed to readily enter the brain after i.v. administration and to be efficiently converted to its glutathione conjugate (MRP1 substrate) in situ. Dynamic PET scan provided the brain time-activity curve after injection of 6-bromo-7-[11C]methylpurine into mice. The efflux rate of the substrate was kinetically estd. to be 1.4 h-1 with high precision. Moreover, knockout of Mrp1 gene caused approx. a 90% redn. of the efflux rate, compared with wild-type mice. In conclusion, our method allows noninvasive and quant. assessment for MRP1 function in the living brain.365Okamura, T.; Kikuchi, T.; Fukushi, K.; Arano, Y.; Irie, T. A novel noninvasive method for assessing glutathione-conjugate efflux systems in the brain. Bioorg. Med. Chem. 2007, 15, 3127– 3133, DOI: 10.1016/j.bmc.2007.02.045[Crossref], [PubMed], [CAS], Google Scholar365https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjslGntL4%253D&md5=b23e187ad43e3205bd8522757b0a4983A novel noninvasive method for assessing glutathione-conjugate efflux systems in the brainOkamura, Toshimitsu; Kikuchi, Tatsuya; Fukushi, Kiyoshi; Arano, Yasushi; Irie, ToshiakiBioorganic & Medicinal Chemistry (2007), 15 (9), 3127-3133CODEN: BMECEP; ISSN:0968-0896. (Elsevier Ltd.)Brain efflux systems export such conjugated metabolites as glutathione (GSH) and glucuronate conjugates, generated by the detoxification process, from the brain and serve to protect the brain from harmful metabolites. The intracerebral injection of a radiolabeled conjugate is a useful technique to assess brain efflux systems; however, this technique is not applicable to humans. Hence, we devised a novel noninvasive approach for assessing GSH-conjugate efflux systems using positron emission tomog. Here, we investigated whether or not a designed proprobe can deliver its GSH conjugate into the brain. Radiolabeled 6-chloro-7-methylpurine (7m6CP) was designed as the proprobe, and [14C]7m6CP was prepd. by the reaction of 6-chloropurine with [14C]CH3I as a model of [11C]CH3I. The radiochem. yield and purity of [14C]7m6CP were 10-20% and greater than 99%, resp. High brain uptake (0.8% ID/g) at 1 min was obsd., followed by gradual radioactivity clearance from the brain for 5-60 min after the injection of [14C]7m6CP into rats. Anal. of metabolites confirmed that the presence of [14C]7m6CP was hardly obsd., and 80% of the radioactivity was identical to its GSH conjugate for 15-60 min. The brain radioactivity was single-exponentially decreased during the period of 15-60 min post-injection of [14C]7m6CP, and the first-order efflux rate const. of the conjugate, estd. from the slope, was 0.0253 min-1. These results showed that (1) [14C]7m6CP readily entered the brain, (2) it efficiently and specifically transformed to the GSH conjugate within the brain, and (3) after [14C]7m6CP disappearance, the clearance of radioactivity represented the only efflux of GSH conjugate. We conclude that 7m6CP can deliver the GSH conjugate into the brain and would be useful for assessing GSH-conjugate efflux systems noninvasively.366Okamura, T.; Kikuchi, T.; Fukushi, K.; Irie, T. Reactivity of 6-halopurine analogs with glutathione as a radiotracer for assessing function of multidrug resistance-associated protein 1. J. Med. Chem. 2009, 52, 7284– 7288, DOI: 10.1021/jm901332c[ACS Full Text], [CAS], Google Scholar366https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlWmurfE&md5=7ebcc20c09d144e2e5df3c8b595362b7Reactivity of 6-Halopurine Analogs with Glutathione as a Radiotracer for Assessing Function of Multidrug Resistance-Associated Protein 1Okamura, Toshimitsu; Kikuchi, Tatsuya; Fukushi, Kiyoshi; Irie, ToshiakiJournal of Medicinal Chemistry (2009), 52 (22), 7284-7288CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)6-Bromo-7-[11C]methylpurine is reported to react with glutathione via glutathione S-transferases in the brain and to be converted into a substrate for multidrug resistance-assocd. protein 1 (MRP1), an efflux pump. The compd. with a rapid conversion rate allows quant. assessment of MRP1 function, but this rate is probably susceptible to interspecies differences. Hence, for application to different species, including humans, it is necessary to adjust the conversion rate by modifying the chem. structure. We therefore designed 6-halo-9-(or 7)-[ 14C]methylpurine (halogen: F, Cl, Br, or I), and evaluated them in vitro with respect to enzymic reactivity with glutathione using brain homogenates from the mouse, rat, or monkey. There was a marked difference in reactivity between these species. Changes in the position of the Me group and halogen on N-methyl-6-halopurine provided various compds. possessing wide-ranging reactivity with glutathione. In conclusion, the adjustment of reactivity of 6-bromo-7-[11C]methylpurine may allow assessment of MRP1 function in the brain in various species.367Liu, J.; Robins, M. J. S(N)Ar displacements with 6-(fluoro, chloro, bromo, iodo, and alkylsulfonyl)purine nucleosides: synthesis, kinetics, and mechanism1. J. Am. Chem. Soc. 2007, 129, 5962– 5968, DOI: 10.1021/ja070021u[ACS Full Text], [CAS], Google Scholar367https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXkt1ShsL0%253D&md5=2da27fced83f8cc103006f056109de56SNAr Displacements with 6-(Fluoro, Chloro, Bromo, Iodo, and Alkylsulfonyl)purine Nucleosides: Synthesis, Kinetics, and MechanismLiu, Jiangqiong; Robins, Morris J.Journal of the American Chemical Society (2007), 129 (18), 5962-5968CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)SNAr reactions with 6-(fluoro, chloro, bromo, iodo, and alkylsulfonyl)purine nucleosides and nitrogen, oxygen, and sulfur nucleophiles were studied. Pseudo-first-order kinetics were measured with 6-halopurine compds., and comparative reactivities were detd. vs. a 6-(alkylsulfonyl)purine nucleoside. The displacement reactivity order was: F > Br > Cl > I (with BuNH2/MeCN), F > Cl ≈ Br > I (with MeOH/1,8-diazabicyclo[5.4.0]undec-7-ene/MeCN), and F > Br > I > Cl [with KSCOCH3/DMSO]. The order of reactivity with a weakly basic arylamine (aniline) was: I > Br > Cl » F (with 5 equiv of aniline in MeCN at 70 °C). However, those reactions with aniline were auto-catalytic and had significant induction periods (∼50 min for the iodo compd. and ∼6 h for the fluoro analog). Addn. of trifluoroacetic acid (TFA) eliminated the induction period, and the order then was F > I > Br > Cl (with 5 equiv of aniline and 2 equiv of TFA in MeCN at 50 °C). The 6-(alkylsulfonyl)purine nucleoside analog was more reactive than the 6-fluoropurine compd. with both MeOH/DBU/MeCN and iPentSH/DBU/MeCN and was more reactive than the Cl, Br, and I compds. with BuNH2 and aniline/TFA. Titrn. of the 6-halopurine nucleosides in CDCl3 with TFA showed progressive downfield 1H NMR chem. shifts for H8 (larger) and H2 (smaller). The major site of protonation as N7 for both the 6-fluoro and 6-bromo analogs was confirmed by large upfield shifts (∼16 ppm) of the 15N NMR signal for N7 upon addn. of TFA (1.6 equiv). Mechanistic considerations and resoln. of prior conflicting results are presented.368Bradamante, S.; Pagani, G. A. Benzyl and heteroarylmethyl carbanions: structure and substituent effects. Adv. Carbanion Chem. 1997, 2, 189– 263, DOI: 10.1016/S1068-7394(96)80006-6369Abbotto, A.; Alanzo, V.; Bradamante, S.; Pagani, G. A. Preparation of heteroaryl phenylmethanes and a 13C and 15N NMR spectroscopic study of their conjugate carbanions. Rotational isomerism and charge maps of the anions and ranking of the charge demands of the heterocycles. J. Chem. Soc., Perkin Trans. 2 1991, 481– 488, DOI: 10.1039/p29910000481[Crossref], [CAS], Google Scholar369https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXktVOqtb0%253D&md5=8e6fc4be2b709d75303979f360d42a8dPreparation of heteroaryl phenylmethanes and a carbon-13 and nitrogen-15 NMR spectroscopic study of their conjugate carbanions. Rotational isomerism and charge maps of the anions and ranking of the charge demands of the heterocyclesAbbotto, Alessandro; Alanzo, Vito; Bradamante, Silvia; Pagani, Giorgio A.Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry (1972-1999) (1991), (4), 481-8CODEN: JCPKBH; ISSN:0300-9580.2-Benzylpyridazine, 4-benzylpyrimidine, 2-benzylpyrimidine and 2-benzylpyrazine have been prepd. in order to study their 13C and 15N spectra and those of their conjugate carbanions. These systems are aza-homologs of the previously reported benzylpyridines and have been considered in order to evaluate the effect of aza-substitution upon rotational isomerism and charge maps in the anions. Two synthetic approaches have been followed: (i) decarboxylation of α-(heteroaryl)phenylacetic acids, in turn obtained by nucleophilic substitution of phenylacetonitrile anion on the pertinent haloazine (or a correspondingly available deriv.); (ii) by nucleophilic substitution of benzyl(tributylphosphonium)ylide on the pertinent haloazine. The 13C and 15N NMR data for the conjugate carbanions indicate that, at room temp., there is slow rotation about the bond between the carbanionic carbon and the carbon atom of the heterocycle: this generates geometrical isomerism in the anions. The NMR data are treated with the π-charge-shift equations to obtain the local variations of the π-electron d. In the anions, from the fraction of π-charge transferred to the heterocycles, it is possible to obtain the charge demands of the heterocycles and thus rank them on the same scale as primary org. functionalities. 4-Pyrimidyl is the strongest electron-withdrawing heterocyclic residue, comparable with the acetyl group.370Shirasaka, T.; Murakami, K.; Ford, H., Jr.; Kelley, J. A.; Yoshioka, H.; Kojima, E.; Aoki, S.; Broder, S.; Mitsuya, H. Lipophilic halogenated congeners of 2’,3′-dideoxypurine nucleosides active against human immunodeficiency virus in vitro. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 9426– 9430, DOI: 10.1073/pnas.87.23.9426[Crossref], [PubMed], [CAS], Google Scholar370https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXpsVKguw%253D%253D&md5=1c73ce3c59782599733ee83429b9333aLipophilic halogenated congeners of 2',3'-dideoxypurine nucleosides active against human immunodeficiency virus in vitroShirasaka, Takuma; Murakami, Kunichika; Ford, Harry, Jr.; Kelley, James A.; Yoshioka, Hidetoshi; Kojima, Eiji; Aoki, Shizuko; Broder, Samuel; Mitsuya, HiroakiProceedings of the National Academy of Sciences of the United States of America (1990), 87 (23), 9426-30CODEN: PNASA6; ISSN:0027-8424.Four 2-amino-6-halo- and four 6-halo-2',3'-dideoxypurine ribofuranosides (ddPs) I (R1 = Cl, F, Br, I, SH; R2 = H or NH2) tested for in vitro activity to suppress the infectivity, cytopathic effect, Gag protein expression, and DNA synthesis of human immunodeficiency virus (HIV). The comparative order of in vitro anti-HIV activity of the eight 6-halo-ddPs was as follows: 2-amino-6-fluoro, 2-amino-6-chloro, 6-fluoro > 2-amino-6-bromo > 2-amino-6-iodo, 6-chloro > 6-bromo > 6-iodo. 2-Amino-6-fluoro-, 2-amino-6-chloro-, and 6-fluoro-ddPs showed a potent activity against HIV comparable to that of 2',3'-dideoxyinosine (ddI) or 2',3'-dideoxyguanosine (ddG) and completely blocked the infectivity of HIV without affecting the growth of target cells. The lipophilciity order was as follows: 2-amino-6-iodo > 2-amino-6-bromo > 2-amino-6-chloro > 2-amino-6-fluoro » ddG > ddI. All eight 6-halo-ddPs were substrates for adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4). The relative rates of hydrolysis by ADA were as follows: ddA, 2-amino-6-fluoro » 2-amino-6-chloro, 2-amino-6-bromo > 2-amino-6-iodo. Taken together, these compds. may represent an addnl. class of lipophilic prodrugs for ddI and ddG and may also provide a strategy for endowing therapeutic purine nucleosides with desirable lipophilicity.371Morgan, M. E.; Chi, S. C.; Murakami, K.; Mitsuya, H.; Anderson, B. D. Central nervous system targeting of 2’,3′-dideoxyinosine via adenosine deaminase-activated 6-halo-dideoxypurine prodrugs. Antimicrob. Agents Chemother. 1992, 36, 2156– 2165, DOI: 10.1128/AAC.36.10.2156[Crossref], [PubMed], [CAS], Google Scholar371https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XmtF2qsbw%253D&md5=48627e036850f263d2013982faf0dacfCentral nervous system targeting of 2',3'-dideoxyinosine via adenosine deaminase-activated 6-halo-dideoxypurine prodrugsMorgan, M. E.; Chi, S. C.; Murakami, K.; Mitsuya, H.; Anderson, B. D.Antimicrobial Agents and Chemotherapy (1992), 36 (10), 2156-65CODEN: AMACCQ; ISSN:0066-4804.AIDS dementia complex is a neurol. disorder, characterized by increasingly severe cognitive, behavioral, and motor impairment, which is assocd. with human immunodeficiency virus (HIV) infection in the central nervous system (CNS). Many of the dideoxynucleosides effective systemically in the treatment of HIV infections, such as 2',3'-dideoxyinosine (ddI), exhibit limited penetration into the CNS and limited or variable effectiveness in reversing the symptoms of AIDS dementia. Thus, approaches for increasing the CNS uptake of ddI and other dideoxynucleosides are needed. The CNS uptake of a series of 6-halo-2',3'-dideoxypurine ribofuranosides (6-halo-ddPs) previously shown to be active against HIV because of their conversion to ddI through the action of adenosine deaminase was examd. in rats. In vitro studies in rat blood and brain tissue homogenate suggested a favorable selectivity for bioconversion in brain tissue, but with bioconversion half-lives varying widely within the series. In vivo infusions of 6-chloro-ddP (6-Cl-ddP), 6-bromo-ddP (6-Br-ddP), and 6-iodo-ddP (6-I-ddP) resulted in significant increases (20- to 34-fold) in the ddI concn. ratios in brain parenchyma/plasma when compared with those after an infusion of ddI alone. Abs. concns. of ddI in brain parenchyma were increased 10- and 4-fold, resp., following 30-min infusions of 6-Cl-ddP or 6-Br-ddP, but were 2.4 fold lower after an infusion of 6-I-ddP relative to that after a control infusion of ddI. Detailed studies of the plasma pharmacokinetics, CNS uptake kinetics, and bioconversion of 6-Cl-ddP were conducted to compare in vivo transport and bioconversion parameters with those predicted from in vitro measurements and to rationalize the efficiency of CNS delivery of ddI from 6-Cl-ddP. The results show that increased lipophilicity alone does not ensure that a given prodrug will deliver higher levels of a parent compd. to the CNS. Both the selectivity and abs. rate of bioconversion in the brain are important factors.372Kim, D.-K.; Kim, J.-K.; Chae, Y.-B. Design and synthesis of 6-fluoropurine acyclonucleosides: potential prodrugs of acyclovir and ganciclovir. Bioorg. Med. Chem. Lett. 1994, 4, 1309– 1312, DOI: 10.1016/S0960-894X(01)80350-4[Crossref], [CAS], Google Scholar372https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXmtVGrtL0%253D&md5=9478dbb3766d5fcb5c0fa8f977c15e9bDesign and synthesis of 6-fluoropurine acyclonucleosides: potential prodrugs of acyclovir and ganciclovirKim, Dae Kee; Kim, Hee Kap; Chae, Young BokBioorganic & Medicinal Chemistry Letters (1994), 4 (11), 1309-12CODEN: BMCLE8; ISSN:0960-894X.6-Fluoropurine acyclic nucleosides I (R = H, CH2OH, R1 = F) (II) have been prepd. as potential prodrugs of acyclovir and ganciclovir. It has been found that II are 11.6 and 7.6 times more efficiently metabolized to acyclovir and ganciclovir by adenosine deaminase than the corresponding 6-aminopurine acyclonucleosides I (R = H, CH2OH, R1 = NH2).373Kim, D. K.; Lee, N.; Im, G. J.; Kim, H. T.; Kim, K. H. Synthesis and evaluation of 2-amino-6-fluoro-9-(2-hydroxyethoxymethyl)purine esters as potential prodrugs of acyclovir. Bioorg. Med. Chem. 1998, 6, 2525– 2530, DOI: 10.1016/S0968-0896(98)80026-6[Crossref], [PubMed], [CAS], Google Scholar373https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXjvVKhsw%253D%253D&md5=417b0d155bab9c6fe31d8871160d87c6Synthesis and evaluation of 2-amino-6-fluoro-9-(2-hydroxyethoxymethyl)purine esters as potential prodrugs of acyclovirKim, Dae-Kee; Lee, Namkyu; Im, Guang-Jin; Kim, Hun-Taek; Kim, Key H.Bioorganic & Medicinal Chemistry (1998), 6 (12), 2525-2530CODEN: BMECEP; ISSN:0968-0896. (Elsevier Science Ltd.)2-Amino-6-fluoro-9-(2-hydroxyethoxymethyl)purine (I) and its ester derivs. were synthesized as potential prodrugs of acyclovir, and were evaluated for their oral acyclovir bioavailability in rats and in vivo antiviral efficacy in HSV-1-infected mice. Treatment of 2-amino-6-chloro-9-(2-hydroxyethoxymethyl)purine with trimethylamine in THF/DMF (4:1) followed by a reaction of the resulting trimethylammonium chloride salt with KF in DMF gave I in 78% yield. Esterification of I with an appropriate acid anhydride (Ac2O, (EtCO)2O, (n-PrCO)2O, or (i-PrCO)2O) in DMF in the presence of a catalytic amt. of DMAP at room temp. produced the esters in 90-98% yields. Of the prodrugs tested in rats, the isobutyrate achieved the highest mean urinary recovery of acyclovir (51%) that is 5.7-fold higher than that of acyclovir (9%) and comparable to that of valacyclovir (50%). The prodrug isobutyrate protected dose-dependently the mortality of HSV-1-infected mice, and the group treated with the isobutyrate at a dose of 400 mg/kg showed the longest mean survival day (14.6 ± 3.1 days) (mean ± S.D.).374Kim, D. K.; Chang, K.; Im, G. J.; Kim, H. T.; Lee, N.; Kim, K. H. Synthesis and evaluation of 2-amino-9-(1, 3-dihydroxy-2-propoxymethyl)- 6-fluoropurine mono- and diesters as potential prodrugs of ganciclovir. J. Med. Chem. 1999, 42, 324– 328, DOI: 10.1021/jm980321+[ACS Full Text], [CAS], Google Scholar374https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXos1Gi&md5=dbca4a20f373e22a7b32f86879198c96Synthesis and evaluation of 2-amino-9-(1,3-dihydroxy-2-propoxymethyl)- 6-fluoropurine mono- and diesters as potential prodrugs of ganciclovirKim, Dae-Kee; Chang, Kieyoung; Im, Guang-Jin; Kim, Hun-Taek; Lee, Namkyu; Kim, Key H.Journal of Medicinal Chemistry (1999), 42 (2), 324-328CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of 2-amino-9-(1,3-dihydroxy-2-propoxymethyl)-6-fluoropurine mono- and diesters, I [R = COMe, COEt, CO(CH2)2Me, COCHMe2, H, R1 = COMe, COEt, CO(CH2)2Me, COCHMe2], were synthesized as potential prodrugs of ganciclovir and evaluated for their oral ganciclovir bioavailability in rats. Treatment of 2-amino-6-chloro-9-(1,3-dihydroxy-2-propoxymethyl)purine with Me3N in DMF/THF (1/4) followed by the reaction of the resulting trimethylammonium chloride salt with KF in DMF gave 2-amino-9-(1,3-dihydroxy-2-propoxymethyl)-6-fluoropurine (II) in 83% yield. Esterification of II with an appropriate acid anhydride (Ac2O, (EtCO)2O, (n-PrCO)2O, or (i-PrCO)2O) in DMF in the presence of a catalytic amt. of DMAP produced the diesters in 92-98% yields and the monoesters in 37-44% yields. Of the prodrugs tested in rats, the monoisobutyrate achieved the highest ganciclovir bioavailability (45%) that is 15-fold higher than that from ganciclovir (3%), followed in order by the diisobutyrate (42%), the diacetate (41%), the monobutyrate (41%), the monopropionate (39%), the dipropionate (35%), the dibutyrate (35%), and the monoacetate (29%). The prodrugs I were found to be quite stable at pH 6.0 (t1/2 = >29 days), 7.4 (t1/2 = >7 days), and 8.0 (t1/2 = >2 days) but had relatively short half-lives at pH 1.2 (t1/2 = 60-83 min).375Kim, D. K.; Lee, N.; Kim, H. T.; Im, G. J.; Kim, K. H. Synthesis and evaluation of 2-amino-6-fluoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine mono- and diesters as potential prodrugs of penciclovir. Bioorg. Med. Chem. 1999, 7, 565– 570, DOI: 10.1016/S0968-0896(98)00263-6[Crossref], [PubMed], [CAS], Google Scholar375https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXitlGktbk%253D&md5=6620297f8c4cbc0724a273fa7197761fSynthesis and Evaluation of 2-amino-6-fluoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine mono- and diesters as potential prodrugs of penciclovirKim, Dae-Kee; Lee, Namkyu; Kim, Hun-Taek; Im, Guang-Jin; Kim, Key H.Bioorganic & Medicinal Chemistry (1999), 7 (3), 565-570CODEN: BMECEP; ISSN:0968-0896. (Elsevier Science Ltd.)2-Amino-6-fluoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine, and its mono- and diesters were prepd. and evaluated for their potential as prodrugs of penciclovir. Treatment of 2-amino-6-chloro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine with trimethylamine in THF followed by a reaction of the resulting trimethylammonium chloride salt with KF in DMF afforded 2-amino-6-fluoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine in 80% yield. Esterification with an appropriate acid anhydride [Ac2O, (EtCO)2O, (n-PrCO)2O, or (i-PrCO)2O] in DMF in the presence of a catalytic amt. of DMAP produced the mono-esters in 42-45% yields and diesters in 87-99% yields. Of the prodrugs tested in rats, the mono-isobutyrate was the most efficiently absorbed and metabolized, showing the mean max. total concn. of penciclovir (5.5 μg/mL) and 2-Amino-6-fluoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine (10.8 μg/mL) in the blood was much higher than the mean max. concn. of penciclovir (11.5 μg/mL) from famciclovir. However, the mean concns. of penciclovir from the mono-isobutyrate were lower than those from famciclovir because of the limited conversion of a major metabolite to penciclovir by adenosine deaminase.376Ouvry, G.; Clary, L.; Tomas, L.; Aurelly, M.; Bonnary, L.; Borde, M.; Bouix-Peter, C.; Chantalat, L.; Defoin-Platel, C.; Deret, S.; Forissier, M.; Harris, C. S.; Isabet, T.; Lamy, L.; Luzy, A.-P.; Pascau, J.; Soulet, C.; Taddei, A.; Taquet, N.; Thoreau, E.; Varvier, E.; Vial, E.; Hennequin, L. F. Impact of minor structural modifications on properties of a series of mTOR inhibitors. ACS Med. Chem. Lett. 2019, 10, 1561– 1567, DOI: 10.1021/acsmedchemlett.9b00401[ACS Full Text], [CAS], Google Scholar376https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvFSru7bI&md5=aa5bf253d605bbc6bfe1dfbf43de24b8Impact of Minor Structural Modifications on Properties of a Series of mTOR InhibitorsOuvry, Gilles; Clary, Laurence; Tomas, Loic; Aurelly, Michele; Bonnary, Laetitia; Borde, Emilie; Bouix-Peter, Claire; Chantalat, Laurent; Defoin-Platel, Claire; Deret, Sophie; Forissier, Mathieu; Harris, Craig S.; Isabet, Tatiana; Lamy, Laurent; Luzy, Anne-Pascale; Pascau, Jonathan; Soulet, Catherine; Taddei, Alessandro; Taquet, Nathalie; Thoreau, Etienne; Varvier, Emeric; Vial, Emmanuel; Hennequin, Laurent F.ACS Medicinal Chemistry Letters (2019), 10 (11), 1561-1567CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)Minor structural modifications (sometimes single atom changes) can have a dramatic impact on the properties of compds. This is illustrated here on structures related to known mTOR inhibitor Sapanisertib. Subtle changes in the hinge binder lead to strikingly different overall profiles with changes in phys. properties, metab., and kinase selectivity.377Boehringer, M.; Fischer, H.; Hennig, M.; Hunziker, D.; Huwyler, J.; Kuhn, B.; Loeffler, B. M.; Luebbers, T.; Mattei, P.; Narquizian, R.; Sebokova, E.; Sprecher, U.; Wessel, H. P. Aryl- and heteroaryl-substituted aminobenzo[a]quinolizines as dipeptidyl peptidase IV inhibitors. Bioorg. Med. Chem. Lett. 2010, 20, 1106– 1108, DOI: 10.1016/j.bmcl.2009.12.025[Crossref], [PubMed], [CAS], Google Scholar377https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVGrsb4%253D&md5=b1d3c1eaa70297eab5ca1abfcd1d7e5dAryl- and heteroaryl-substituted aminobenzo[a]quinolizines as dipeptidyl peptidase IV inhibitorsBoehringer, Markus; Fischer, Holger; Hennig, Michael; Hunziker, Daniel; Huwyler, Joerg; Kuhn, Bernd; Loeffler, Bernd M.; Luebbers, Thomas; Mattei, Patrizio; Narquizian, Robert; Sebokova, Elena; Sprecher, Urs; Wessel, Hans PeterBioorganic & Medicinal Chemistry Letters (2010), 20 (3), 1106-1108CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Synthesis and SAR are described for a structurally distinct class of DPP-IV inhibitors based on aminobenzo[a]quinolizines bearing (hetero-)arom. substituents in the S1 specificity pocket. The m-(fluoromethyl)-Ph deriv. (S,S,S)-I possesses the best fit in the S1 pocket. However, (S,S,S)-II, bearing a more hydrophilic 5-methyl-pyridin-2-yl residue as substituent for the S1 pocket, displays excellent in vivo activity and superior drug-like properties.378Mattei, P.; Boehringer, M.; Di Giorgio, P.; Fischer, H.; Hennig, M.; Huwyler, J.; Kocer, B.; Kuhn, B.; Loeffler, B. M.; Macdonald, A.; Narquizian, R.; Rauber, E.; Sebokova, E.; Sprecher, U. Discovery of carmegliptin: a potent and long-acting dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. Bioorg. Med. Chem. Lett. 2010, 20, 1109– 1113, DOI: 10.1016/j.bmcl.2009.12.024[Crossref], [PubMed], [CAS], Google Scholar378https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVGrsb8%253D&md5=448a62baa339f33ea89a9f7d862a7c25Discovery of carmegliptin: A potent and long-acting dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetesMattei, Patrizio; Boehringer, Markus; Di Giorgio, Patrick; Fischer, Holger; Hennig, Michael; Huwyler, Joerg; Kocer, Buelent; Kuhn, Bernd; Loeffler, Bernd M.; MacDonald, Alexander; Narquizian, Robert; Rauber, Etienne; Sebokova, Elena; Sprecher, UrsBioorganic & Medicinal Chemistry Letters (2010), 20 (3), 1109-1113CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Design, synthesis, and SAR are described for a class of DPP-IV inhibitors based on aminobenzo[a]quinolizines with non-arom. substituents in the S1 specificity pocket. One representative thereof, carmegliptin (I), was chosen for clin. development. The X-ray structure in complex of I with the enzyme and early efficacy data in animal models of type 2 diabetes are also presented.379Kuhlmann, O.; Paehler, A.; Weick, I.; Funk, C.; Pantze, M.; Zell, M.; Timm, U. Pharmacokinetics and metabolism of the dipeptidyl peptidase IV inhibitor carmegliptin in rats, dogs, and monkeys. Xenobiotica 2010, 40, 840– 852, DOI: 10.3109/00498254.2010.519406[Crossref], [PubMed], [CAS], Google Scholar379https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3cbls1ehsg%253D%253D&md5=41599b82f0246f119097a818d0a1c7abPharmacokinetics and metabolism of the dipeptidyl peptidase IV inhibitor carmegliptin in rats, dogs, and monkeysKuhlmann Olaf; Paehler Axel; Weick Idelette; Funk Christoph; Pantze Michael; Zell Manfred; Timm UweXenobiotica; the fate of foreign compounds in biological systems (2010), 40 (12), 840-52 ISSN:.The pharmacokinetics and excretion of carmegliptin, a novel dipeptidyl peptidase IV inhibitor, were examined in rats, dogs, and cynomolgus monkeys. Carmegliptin exhibited a moderate clearance, extensive tissue distribution, and a variable oral bioavailability of 28-174%. Due to saturation of intestinal active secretion, the area under the plasma concentration-time curve (AUC) in dogs and monkeys increased in a more than dose-proportional manner over an oral dose range of 2.5-10 mg/kg. Following oral administration of [(14)C]carmegliptin at 3 mg/kg, > 94% of the radioactive dose was recovered in 72-h post-dose from Wistar rats and Beagle dogs. Virtually, the entire administered radioactive dose was excreted unchanged in urine, intestinal lumen, and bile. Approximately 36%, 29%, and 19% of the dose were excreted by respective routes. Consistently, in vitro, carmegliptin was highly resistant to hepatic metabolism in all species tested. Based on in vitro studies, carmegliptin is a good substrate for Mdr1/MDR1. Breast cancer resistance protein (Bcrp) is not expected to be involved in the transport of carmegliptin since in vitro carmegliptin was not significantly transported by this transporter. The very high extravascular distribution of carmegliptin in the intestinal tissues, as demonstrated in Wistar rats and Beagle dogs, could play a significant role in its therapeutic effect.380Shetty, H. U.; Zoghbi, S. S.; Simeon, F. G.; Liow, J. S.; Brown, A. K.; Kannan, P.; Innis, R. B.; Pike, V. W. Radiodefluorination of 3-fluoro-5-(2-(2-[18F](fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile ([18F]SP203), a radioligand for imaging brain metabotropic glutamate subtype-5 receptors with positron emission tomography, occurs by glutathionylation in rat brain. J. Pharmacol. Exp. Ther. 2008, 327, 727– 735, DOI: 10.1124/jpet.108.143347[Crossref], [PubMed], [CAS], Google Scholar380https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVCjtLvN&md5=7cc78a76561a92270f956ca354d259efRadiodefluorination of 3-fluoro-5-(2-(2-[18F](fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile ([18F]SP203), a radioligand for imaging brain metabotropic glutamate subtype-5 receptors with positron emission tomography, occurs by glutathionylation in rat brainShetty, H. Umesha; Zoghbi, Sami S.; Simeon, Fabrice G.; Liow, Jeih-San; Brown, Amira K.; Kannan, Pavitra; Innis, Robert B.; Pike, Victor W.Journal of Pharmacology and Experimental Therapeutics (2008), 327 (3), 727-735CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)Metabotropic glutamate subtype-5 receptors (mGluR5) are implicated in several neuropsychiatric disorders. Positron emission tomog. (PET) with a suitable radioligand may enable monitoring of regional brain mGluR5 d. before and during treatments. We have developed a new radioligand, 3-fluoro-5-(2-(2-[18F](fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile ([18F]SP203), for imaging brain mGluR5 in monkey and human. In monkey, radioactivity was obsd. in bone, showing release of [18F]fluoride ion from [18F]SP203. This defluorination was not inhibited by disulfiram, a potent inhibitor of CYP2E1. PET confirmed bone uptake of radioactivity and therefore defluorination of [18F]SP203 in rats. To understand the biochem. basis for defluorination, we administered [18F]SP203 plus SP203 in rats for ex vivo anal. of metabolites. Radio-high-performance liq. chromatog. detected [18F]fluoride ion as a major radiometabolite in both brain ext. and urine. Incubation of [18F]SP203 with brain homogenate also generated this radiometabolite, whereas no metab. was detected in whole blood in vitro. Liq. chromatog.-mass spectrometry anal. of the brain ext. detected m/z 548 and 404 ions, assignable to the [M + H]+ of S-glutathione (SP203Glu) and N-acetyl-S-L-cysteine (SP203Nac) conjugates of SP203, resp. In urine, only the [M + H]+ of SP203Nac was detected. Mass spectrometry/mass spectrometry and multi-stage mass spectrometry analyses of each metabolite yielded product ions consistent with its proposed structure, including the former fluoromethyl group as the site of conjugation. Metabolite structures were confirmed by similar analyses of SP203Glu and SP203Nac, prepd. by glutathione S-transferase reaction and chem. synthesis, resp. Thus, glutathionylation at the 2-fluoromethyl group is responsible for the radiodefluorination of [18F]SP203 in rat. This study provides the first demonstration of glutathione-promoted radiodefluorination of a PET radioligand.381McDonald, I. A.; Nyce, P. L.; Jung, M. J.; Sabol, J. S. Syntheses of dl-2-fluoromethyl-p-tyrosine and dl-2-difluoromethyl-p-tyrosine as potential inhibitors of tyrosine hydroxylase. Tetrahedron Lett. 1991, 32, 887– 890, DOI: 10.1016/S0040-4039(00)92112-9[Crossref], [CAS], Google Scholar381https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXhvVKjtrY%253D&md5=94ea2a823059fc2697e102cefd0486d9Syntheses of DL-2-fluoromethyl-p-tyrosine and DL-2-(difluoromethyl)-p-tyrosine as potential inhibitors of tyrosine hydroxylaseMcDonald, Ian A.; Nyce, Philip L.; Jung, Michel J.; Sabol, Jeffrey S.Tetrahedron Letters (1991), 32 (7), 887-90CODEN: TELEAY; ISSN:0040-4039.The title compds. I (R = H and F) were prepd. from o-xylene II and benzoate III, resp. I (R = H) was obtained from II in 11 steps; a key step was the free radical bromination of tyrosine IV (Boc = Me3CO2C, R1 = H) with NBS followed by treatment with AgF to give IV (R1 = F). I (R = F) was prepd. from III in 11 steps. I (R = H, F) were partially characterized as competitive inhibitors of purified bovine adrenal tyrosine hydroxylase.382Robinson, C. Metyrosine. Drugs Today 1980, 16, 343– 348[CAS], Google Scholar382https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXntFagsA%253D%253D&md5=dac351a91b456574062cfc5f851e82f8MetyrosineRobinson, C.Drugs of Today (1980), 16 (10), 343-8CODEN: MDACAP; ISSN:0025-7656.The present status of the drug metyrosine (I) [672-87-7] is reviewed; 10 refs.383Woolridge, E. M.; Rokita, S. E. 6-(Difluoromethyl)tryptophan as a probe for substrate activation during the catalysis of tryptophanase. Biochemistry 1991, 30, 1852– 1857, DOI: 10.1021/bi00221a018[ACS Full Text], [CAS], Google Scholar383https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXnslKgtw%253D%253D&md5=672d34bfbe9d3dcd4d7f7772565472056-(Difluoromethyl)tryptophan as a probe for substrate activation during the catalysis of tryptophanaseWoolridge, Elisa M.; Rokita, Steven E.Biochemistry (1991), 30 (7), 1852-7CODEN: BICHAW; ISSN:0006-2960.A substrate analog, 6-(difluoromethyl)tryptophan, was developed and characterized for mechanistic investigation of tryptophanase of Escherichia coli. The utility of this deriv. was based on its ability to partition between F- elimination and C-C bond scission during tryptophan metab. The nonenzymic hydrolysis to 6-formyltryptophan occurred slowly under neutral conditions with a 1st-order rate const. of 0.0039 min-1. This process, however, was accelerated by 104-fold upon deprotonation of the indolyl N-1 atom at high pH. Tryptophanase did not detectably facilitate this hydrolysis reaction, since no protein-dependent conversion of the difluoromethyl group was detected. Instead, the enzyme accepted the fluorinated species as an analog of tryptophan and catalyzed the corresponding formation of 6-(difluoromethyl)indole, pyruvate, and NH4+. Anionic intermediates are therefore not expected to form during the catalytic activation of the indolyl moiety. Instead, arom. protonation likely promotes the release of indole during enzymic degrdn. of tryptophan.384Kai, H.; Hinou, H.; Nishimura, S. Aglycone-focused randomization of 2-difluoromethylphenyl-type sialoside suicide substrates for neuraminidases. Bioorg. Med. Chem. 2012, 20, 2739– 2746, DOI: 10.1016/j.bmc.2012.02.001[Crossref], [PubMed], [CAS], Google Scholar384https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xjs1Oqtbg%253D&md5=935f17620b75bcf8fffdbd1eee849ab7Aglycone-focused randomization of 2-difluoromethylphenyl-type sialoside suicide substrates for neuraminidasesKai, Hirokazu; Hinou, Hiroshi; Nishimura, Shin-IchiroBioorganic & Medicinal Chemistry (2012), 20 (8), 2739-2746CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)A selective and potent inhibitor of neuraminidases, a hydrolase that is responsible for processing sialylated glycoconjugates, is a promising drug candidate for various infective diseases. The current study demonstrates that the use of an aglycon-focused library of 2-difluoromethylphenyl α-sialosides is an effective technique to find potent and selective mechanism-based labeling reagents for neuraminidases. The focused library was constructed from a 4-azide-2-difluoromethylphenyl sialoside (2) and an alkyne-terminated compd. library by a click reaction. The focused library showed different inhibition patterns for two neuraminidases, Vibrio cholerae neuraminidase (VCNA) and human neuraminidase 2 (hNeu2), and the most potent inhibitors for each neuraminidase were selected. A kinetic anal. of the selected inhibitors demonstrated that the modification of the aglycon moiety improved the KI value with little change in the t1/2 value of the enzyme activity relative to the basic skeleton (2).385Kurogochi, M.; Nishimura, S.; Lee, Y. C. Mechanism-based fluorescent labeling of β-galactosidases. An efficient method in proteomics for glycoside hydrolases. J. Biol. Chem. 2004, 279, 44704– 44712, DOI: 10.1074/jbc.M401718200[Crossref], [PubMed], [CAS], Google Scholar385https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXosFymtLw%253D&md5=110f679fc05568ebe1bbac2a3db35bfcMechanism-based Fluorescent Labeling of β-Galactosidases: An Efficient Method in Proteomics for Glycoside HydrolasesKurogochi, Masaki; Nishimura, Shin-Ichiro; Lee, Yuan ChuanJournal of Biological Chemistry (2004), 279 (43), 44704-44712CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)(4-N-5-Dimethylaminonaphthalene-1-sulfonyl-2-difluoromethylphenyl)-β-D-galactopyranoside was synthesized and successfully tested on β-galactosidases from Xanthomonas manihotis, Escherichia coli, and Bacillus circulans for the rapid identification of the catalytic site. Reaction of the irreversible inhibitor with enzymes proceeded to afford a fluorescence-labeled protein suitable for further high throughput characterization by using antidansyl antibody and matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF). Specific probing by a fluorescent aglycon greatly facilitated identification of the labeled peptide fragments from β-galactosidases. It was demonstrated by using X. manihotis β-galactosidase that the Arg-58 residue, which is located within a sequence of 56IPRAYWKD63, was labeled by nucleophilic attack of the guanidinyl group. This sequence including Arg-58 (Leu-46 to Tyr-194) was similar to that (Met-1 to Tyr-151) of Thermus thermophilus A4, which is the first known structure of glycoside hydrolases family 42. A catalytic glutamic acid (Glu-537) of E. coli β-galactosidase was shown to be labeled by the same procedure, suggesting that the modification site with this irreversible substrate might depend both on the nucleophilicity of the amino acids and their spatial arrangement in the individual catalytic cavity. Similarly, a Glu-259 in 257TLEE260 was selectively labeled using B. circulans β-galactosidase, indicating that Glu-259 is one of the nucleophiles in the active site. The present method can be readily extended to other glycosidases and should greatly aid the high throughput proteomics of many glycoside hydrolases showing both retaining- and inverting-type mechanisms.386Tuan, E.; Kirk, K. L. Fluorine reactivity in difluoromethylimidazoles. J. Fluorine Chem. 2006, 127, 980– 982, DOI: 10.1016/j.jfluchem.2006.03.014[Crossref], [CAS], Google Scholar386https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xmt1Gnu70%253D&md5=13ad34b686f5f888805ab05560782ef0Fluorine reactivity in difluoromethylimidazolesTuan, Edward; Kirk, Kenneth L.Journal of Fluorine Chemistry (2006), 127 (7), 980-982CODEN: JFLCAR; ISSN:0022-1139. (Elsevier B.V.)A difluoromethyl substituent attached directly to an imidazole ring is very reactive toward basic hydrolysis. A correlation of rate of fluoride loss with increasing pH is consistent with a mechanism that involves initial ionization of the imidazole NH, formation of an intermediate azafulvene by loss of HF, and reaction of the intermediate with solvent water.387Sakai, T. T.; Santi, D. V. Hydrolysis of hydroxybenzotrifluorides and fluorinated uracil derivatives. A general mechanism for carbon-fluorine bond labilization. J. Med. Chem. 1973, 16, 1079– 1084, DOI: 10.1021/jm00268a003[ACS Full Text], [CAS], Google Scholar387https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3sXlsVejsLc%253D&md5=c458d40df3311cf8e837efa35ab66f33Hydrolysis of hydroxybenzotrifluorides and fluorinated uracil derivatives. General mechanism for carbon-fluorine bond labilizationSakai, Ted T.; Santi, Daniel V.Journal of Medicinal Chemistry (1973), 16 (10), 1079-84CODEN: JMCMAR; ISSN:0022-2623.A general mechanism for C-F bond labilization was presented which may be predictively useful for study of enzyme mechanisms and in design of effective chemotherapeutic drugs. O-hydroxybenzotrifluoride (I) [444-30-4] and p-hydroxybenzotrifluoride [402-45-9] were hydrolyzed at alk. pH to the corresponding hydroxybenzoic acids, and the vinylog 1-trifluoromethyl-2-(4-hydroxyphenyl)ethylene [2338-84-3] was hydrolyzed to p-coumaric acid. 5-Difluoromethyluracil (II) [670-19-9] and 1-methyl-5-difluoromethyluracil [43064-01-3] were hydrolyzed to 5-formyluracil and 1-methyl-5-formyluracil, resp. Kinetics of hydrolysis of the phenolic fluoromethyl compds. suggested participation of the phenolate anions in displacement of fluoride ion to give difluoroquinone methide intermediates, e.g. III. Ability of the compds. to form olefinic intermediates appeared necessary for reaction to occur. A uracil anion probably participated in hydrolysis of the difluorouracil. Conversion of the olefinic intermediates to products presumably involved alternate addn. of nucleophile (or solvent) to the intermediate and elimination of F-.388Peukert, S.; Nunez, J.; He, F.; Dai, M.; Yusuff, N.; DiPesa, A.; Miller-Moslin, K.; Karki, R.; Lagu, B.; Harwell, C.; Zhang, Y.; Bauer, D.; Kelleher, J. F.; Egan, W. A method for estimating the risk of drug-induced phototoxicity and its application to smoothened inhibitors. MedChemComm 2011, 2, 973– 976, DOI: 10.1039/c1md00144b[Crossref], [CAS], Google Scholar388https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1Oqu7nK&md5=af0595f2f52ef672c7a42e32c6dab809A method for estimating the risk of drug-induced phototoxicity and its application to smoothened inhibitorsPeukert, Stefan; Nunez, Jill; He, Feng; Dai, Miao; Yusuff, Naeem; DiPesa, Alan; Miller-Moslin, Karen; Karki, Rajesh; Lagu, Bharat; Harwell, Chris; Zhang, Yalin; Bauer, Daniel; Kelleher, Joseph F.; Egan, WilliamMedChemComm (2011), 2 (10), 973-976CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)The energy difference between the frontier-orbital HOMO-LUMO gaps calcd. for the ground state of marketed oral drugs correlated with their obsd. phototoxicity. This mol. descriptor, together with their maximal molar absorptivity for UV light above 290 nm, can be used to predict phototoxicity risks. This is demonstrated for the phototoxicity mitigation of 1-piperazinyl phthalazines, a class of smoothened inhibitors.389Fournier, J. F.; Bouix-Peter, C.; Duvert, D.; Luzy, A. P.; Ouvry, G. Intrinsic property forecast index (iPFI) as a rule of thumb for medicinal chemists to remove a phototoxicity liability. J. Med. Chem. 2018, 61, 3231– 3236, DOI: 10.1021/acs.jmedchem.8b00075[ACS Full Text], [CAS], Google Scholar389https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXltVGksb8%253D&md5=d9a034f7ce81e84d099e94d26a0573e5Intrinsic Property Forecast Index (iPFI) as a Rule of Thumb for Medicinal Chemists to Remove a Phototoxicity LiabilityFournier, Jean-Francois; Bouix-Peter, Claire; Duvert, Denis; Luzy, Anne-Pascale; Ouvry, GillesJournal of Medicinal Chemistry (2018), 61 (7), 3231-3236CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Phototoxicity occurs when UV irradn. causes otherwise benign compds. to become irritant, sensitizers, or even genotoxic. This toxicity is particularly a concern after topical application and in dermatol. programs where skin irritation can be incompatible with the desired therapeutic outcome. This brief article establishes that the intrinsic property forecast index (iPFI) can be used to evaluate the probability of a compd. being phototoxic and gives medicinal chemists a practical tool to handle this liability.390Schmidt, F.; Wenzel, J.; Halland, N.; Gussregen, S.; Delafoy, L.; Czich, A. Computational investigation of drug phototoxicity: photosafety assessment, photo-toxophore identification, and machine learning. Chem. Res. Toxicol. 2019, 32, 2338– 2352, DOI: 10.1021/acs.chemrestox.9b00338[ACS Full Text], [CAS], Google Scholar390https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvFOrurvP&md5=c181094b301a4c59ef3ac546aada70e0Computational Investigation of Drug Phototoxicity: Photosafety Assessment, Photo-Toxophore Identification, and Machine LearningSchmidt, Friedemann; Wenzel, Jan; Halland, Nis; Guessregen, Stefan; Delafoy, Laure; Czich, AndreasChemical Research in Toxicology (2019), 32 (11), 2338-2352CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)One of the most appreciated capabilities of computational toxicol. is to support the design of pharmaceuticals with reduced toxicol. hazard. To this end, we have strengthened our drug photosafety assessments by applying novel computer models for the anticipation of in vitro phototoxicity and human photosensitization. These models are typically used in pharmaceutical discovery projects as part of the compd. toxicity assessments and compd. optimization methods. To ensure good data quality and aiming at models with global applicability we sep. compiled and curated highly chem. diverse data sets from 3T3 NRU phototoxicity reports (450 compds.) and clin. photosensitization alerts (1419 compds.) which are provided as supplements. The latter data gives rise to a comprehensive list of explanatory fragments for visual guidance, termed phototoxophores, by application of a Bayesian statistics approach. To extend beyond the domain of well sampled fragments we applied machine learning techniques based on explanatory descriptors such as pharmacophoric fingerprints or, more important, accurate electronic energy descriptors. Electronic descriptors were extd. from quantum chem. computations at the d. functional theory (DFT) level. Accurate UV/vis spectral absorption descriptors and pharmacophoric fingerprints turned out to be necessary for predictive computer models, which were both derived from Deep Neural Networks but also the simpler Random Decision Forests approach. Model accuracies of 83-85% could typically be reached for diverse test data sets and other company inhouse data, while model sensitivity (the capability of correctly detecting toxicants) was even better, reaching 86%-90%. Importantly, a computer model-triggered response-map allowed for graphical/chem. interpretability also in the case of previously unknown phototoxophores. The photosafety models described here are currently applied in a prospective manner for the hazard identification, prioritization, and optimization of newly designed mols.391Miolo, G.; Levorato, L.; Gallocchio, F.; Caffieri, S.; Bastianon, C.; Zanoni, R.; Reddi, E. In vitro phototoxicity of phenothiazines: involvement of stable UVA photolysis products formed in aqueous medium. Chem. Res. Toxicol. 2006, 19, 156– 163, DOI: 10.1021/tx0502239[ACS Full Text], [CAS], Google Scholar391https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlWmu7zP&md5=d7376f883194fac567f40cf231d6d34dIn Vitro Phototoxicity of Phenothiazines: Involvement of Stable UVA Photolysis Products Formed in Aqueous MediumMiolo, G.; Levorato, L.; Gallocchio, F.; Caffieri, S.; Bastianon, C.; Zanoni, R.; Reddi, E.Chemical Research in Toxicology (2006), 19 (1), 156-163CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)This paper reports the results of an in vitro evaluation of the phototoxic potential of stable photoproducts formed by UVA photolysis of three phenothiazines, perphenazine, fluphenazine, and thioridazine, in a water environment. Perphenazine gave a single product due to dechlorination. From thioridazine, the two major products formed; the endocyclic sulfoxide and the endocyclic N-oxide in which the 2-SCH3 substituent was replaced by a hydroxy group were tested. From fluphenazine, two products have been examd. as follows: an exocyclic N-piperazine oxide and a carboxylic acid arising from hydrolysis of the 2-CF3 group. The phototoxicity of the isolated photoproducts has been studied to det. their possible involvement in the photosensitizing effects exhibited by the parent drugs, using hemolysis and 3T3 fibroblasts viability as in vitro assays. As fluphenazine, perphenazine, and thioridazine did, some photoproducts proved phototoxic. In particular, the perphenazine dechlorinated photoproduct and the thioridazine N-oxide were found to exert phototoxic properties similar to the parent compds. Therefore, our data suggest that some phenothiazine photoproducts may play a role in the mechanism of photosensitivity of these drugs. Because some of these photoproducts correspond to metabolic products of phenothiazines found in humans, it cannot be ruled out that metabolites of phenothiazines can be phototoxic in vivo.392Caffieri, S.; Miolo, G.; Seraglia, R.; Dalzoppo, D.; Toma, F. M.; Beyersbergen van Henegouwen, G. M. J. Photoaddition of fluphenazine to nucleophiles in peptides and proteins. Possible cause of immune side effects. Chem. Res. Toxicol. 2007, 20, 1470– 1476, DOI: 10.1021/tx700123u[ACS Full Text], [CAS], Google Scholar392https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVKrs73P&md5=1cb0a2a1f2df32520d1c3cf4416bfaf9Photoaddition of Fluphenazine to Nucleophiles in Peptides and Proteins. Possible Cause of Immune Side EffectsCaffieri, Sergio; Miolo, Giorgia; Seraglia, Roberta; Dalzoppo, Daniele; Toma, Francesca M.; Beyersbergen van Henegouwen, Gerard M. J.Chemical Research in Toxicology (2007), 20 (10), 1470-1476CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)By the action of UVA light, fluphenazine reacted with nucleophiles through a mechanism involving defluorination of its trifluoromethyl group, giving rise to carboxylic acid derivs. that were easily detected by electrospray mass spectrometry. This photoreaction took place with alcs., sulphydryls, and amines. When irradn. of fluphenazine was carried out in the presence of an amino acid at pH 7.4, the α-amino group was covalently bound to the drug. With amino acids possessing a further nucleophilic residue on the side chain, such as lysine, tyrosine, and cysteine-but not serine-both groups reacted, resulting in a fluphenazine-amino acid-fluphenazine diadduct. The same occurred with the physiol. peptide glutathione (γ-glutamylcysteinylglycine). By means of MALDI mass spectrometry, it was shown that fluphenazine also covalently bound to peptides and proteins such as calmodulin. This binding may result in the formation of antibodies, ultimately leading to the destruction of the granulocytes and thus suggesting that photoactivation of this drug may play a role in its clin. side effects, such as agranulocytosis.393Lam, M. W.; Young, C. J.; Mabury, S. A. Aqueous photochemical reaction kinetics and transformations of fluoxetine. Environ. Sci. Technol. 2005, 39, 513– 522, DOI: 10.1021/es0494757[ACS Full Text], [CAS], Google Scholar393https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtVCrsbvJ&md5=522617f27e634f4a4a7b80806b1d2d34Aqueous Photochemical Reaction Kinetics and Transformations of FluoxetineLam, Monica W.; Young, Cora J.; Mabury, Scott A.Environmental Science and Technology (2005), 39 (2), 513-522CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)Fluoxetine (FLX) was shown to be photoreactive in sunlit surface water. FLX degraded in deionized water when exposed to simulated sunlight with a half-life of 55.2 ± 3.6 h. Photodegrdn. products were identified by HPLC-UV and liq. chromatog.-tandem mass spectrometry (LC-MS-MS) with electro-spray (ES) ionization. Defluorination of the trifluoromethyl group in FLX and in fluometuron and flutalanil, 2 other compds. contg. this functional group, was suggested to be a common direct photolysis pathway for trifluoromethylated compds.; products resulting from O-dealkylation of FLX were also obsd. The degrdn. rate was faster in synthetic field water where OH- was the likely dominant system oxidant. The bimol. rate const. for the reaction between FLX and OH- was measured as 8.4 ± 0.5 × 109 and 9.6 ± 0.8 × 109 M/s using 2 different methods of competition kinetics. Indirect photodegrdn. reactions could lead to prodn. of hydroxylated and O-dealkylated compds. Although direct photolysis could potentially limit the persistence of FLX in surface water, its degrdn. by indirect photolysis would proceed faster. Thus, the latter process could be important in eliminating FLX in surface water.394Bosca, F.; Cuquerella, M. C.; Marin, M. L.; Miranda, M. A. Photochemistry of 2-hydroxy-4-trifluoromethylbenzoic acid, major metabolite of the photosensitizing platelet antiaggregant drug triflusal. Photochem. Photobiol. 2001, 73, 463– 468, DOI: 10.1562/0031-8655(2001)0730463POHTAM2.0.CO2[Crossref], [PubMed], [CAS], Google Scholar394https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjsFCkt7Y%253D&md5=d540822aa7602ca2767adac042743f08Photochemistry of 2-hydroxy-4-trifluoromethylbenzoic acid, major metabolite of the photosensitizing platelet antiaggregant drug triflusalBosca, F.; Cuquerella, M. C.; Marin, M. L.; Miranda, M. A.Photochemistry and Photobiology (2001), 73 (5), 463-468CODEN: PHCBAP; ISSN:0031-8655. (American Society for Photobiology)Triflusal is a platelet antiaggregant drug with photoallergic side effects. However, it is considered a prodrug since it is metabolized to 2-hydroxy-4-trifluoromethyl-benzoic acid (HTB)-the pharmacol. active form. HTB was found to be photolabile under various conditions. Its major photodegrdn. pathway appears to be the nucleophilic attack at the trifluoromethyl moiety. The involvement of the triplet state in the photodegrdn. has been unequivocally proved by direct detection of this transient in laser flash photolysis and by quenching expts. with oxygen, cyclohexadiene and naphthalene. Finally, the photobinding of HTB to proteins such as bovine serum albumin has been demonstrated using UV-visible (UV-Vis) and fluorescence spectroscopy. Nucleophilic groups present in the protein appear to be responsible for the formation of covalent drug photoadducts, which is the first step involved in the photoallergy shown by triflusal.395Nuin, E.; Perez-Sala, D.; Lhiaubet-Vallet, V.; Andreu, I.; Miranda, M. A. Photosensitivity to triflusal: formation of a photoadduct with ubiquitin demonstrated by photophysical and proteomic techniques. Front. Pharmacol. 2016, 7, 277, DOI: 10.3389/fphar.2016.00277[Crossref], [PubMed], [CAS], Google Scholar395https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFSisrbO&md5=8d2999547cef362049c5dece94c8b49aPhotosensitivity to triflusal: formation of a photoadduct with ubiquitin demonstrated by photophysical and proteomic techniquesNuin, Edurne; Perez-Sala, Dolores; Lhiaubet-Vallet, Virginie; Andreu, Inmaculada; Miranda, Miguel A.Frontiers in Pharmacology (2016), 7 (), 277/1-277/8CODEN: FPRHAU; ISSN:1663-9812. (Frontiers Media S.A.)Triflusal is a platelet aggregation inhibitor chem. related to acetylsalicylic acid, which is used for the prevention and/or treatment of vascular thromboembolisms, which acts as a prodrug. Actually, after oral administration it is absorbed primarily in the small intestine, binds to plasma proteins (99%) and is rapidly biotransformed in the liver into its deacetylated active metabolite 2-hydroxy-4-trifluoromethylbenzoic acid (HTB). In healthy humans, the half-life of triflusal is ca. 0.5 h, whereas for HTB it is ca. 35 h. From a pharmacol. point of view, it is interesting to note that HTB is itself highly active as a platelet anti-aggregant agent. Indeed, studies on the clin. profile of both drug and metabolite have shown no significant differences between them. It has been evidenced that HTB displays ability to induce photoallergy in humans. This phenomenon involves a cell-mediated immune response, which is initiated by covalent binding of a light-activated photosensitizer (or a species derived therefrom) to a protein. In this context, small proteins like ubiquitin could be appropriate models for investigating covalent binding by means of MS/MS and peptide fingerprint anal. In previous work, it was shown that HTB forms covalent photoadducts with isolated lysine. Interestingly, ubiquitin contains seven lysine residues that could be modified by a similar reaction. With this background, the aim of the present work is to explore adduct formation between the triflusal metabolite and ubiquitin as model protein upon sunlight irradn., combining proteomic and photophys. (fluorescence and laser flash photolysis) techniques. Photophys. and proteomic anal. demonstrates monoadduct formation as the major outcome of the reaction. Interestingly, addn. can take place at any of the ε-amino groups of the lysine residues of the protein and involves replacement of the trifluoromethyl moiety with a new amide function. This process can in principle occur with other trifluoroarom. compds. and may be responsible for the appearance of undesired photoallergic side effects.396Chaignon, P.; Cortial, S.; Guerineau, V.; Adeline, M. T.; Giannotti, C.; Fan, G.; Ouazzani, J. Photochemical reactivity of trifluoromethyl aromatic amines: the example of 3,5-diamino-trifluoromethyl-benzene (3,5-DABTF). Photochem. Photobiol. 2005, 81, 1539– 1543, DOI: 10.1562/2005-08-03-RA-637[Crossref], [PubMed], [CAS], Google Scholar396https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlekt7%252FF&md5=ac29f22dfd1188e91fd0dc2dec99bd3cPhotochemical reactivity of trifluoromethyl aromatic amines: The example of 3,5-diamino-trifluoromethyl-benzene (3,5-DABTF)Chaignon, Philippe; Cortial, Sylvie; Guerineau, Vincent; Adeline, Marie-Therese; Giannotti, Charles; Fan, Gerard; Ouazzani, JamalPhotochemistry and Photobiology (2005), 81 (Nov./Dec.), 1539-1543CODEN: PHCBAP; ISSN:0031-8655. (American Society for Photobiology)This work presents the application of an online photoreactor to the detection of 3,5-diamino-trifluoromethyl-benzene (3,5-DABTF) in aq. solns. When irradiated at 310 nm, this compd. is defluorinated to 3,5-diaminobenzoic acid by a nucleophilic substitution of the fluoride by water. Concomitantly, defluorination intermediates polymerize through amide bonds to give dark-colored compds. We take advantage of the photocatalyzed defluorination and the subsequent decrease in pH to develop an original and specific photoreactor. Continuous recording of pH and temp. in the outlet of the reactor by a dual electrode gives us an opportunity to optimize the system. In the photoreactor, 3,5-DABTF is immediately and totally transformed as attested by the rapid drop of the flowing soln. pH from 6.2 to 3.2 and the chromatog. anal. of the outgoing solns. The detection remains effective from 1 to 1000 ppm.397Perez-Ruiz, R.; Molins-Molina, O.; Lence, E.; Gonzalez-Bello, C.; Miranda, M. A.; Jimenez, M. C. Photogeneration of quinone methides as latent electrophiles for lysine targeting. J. Org. Chem. 2018, 83, 13019– 13029, DOI: 10.1021/acs.joc.8b01559[ACS Full Text], [CAS], Google Scholar397https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvVaqtL7O&md5=c8c0eca3cbea34b02c18dbc55d40464cPhotogeneration of Quinone Methides as Latent Electrophiles for Lysine TargetingPerez-Ruiz, Raul; Molins-Molina, Oscar; Lence, Emilio; Gonzalez-Bello, Concepcion; Miranda, Miguel A.; Jimenez, M. ConsueloJournal of Organic Chemistry (2018), 83 (21), 13019-13029CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)Latent electrophiles are nowadays very attractive chem. entities for drug discovery, as they are unreactive unless activated upon binding with the specific target. In this work, the utility of 4-trifluoromethyl phenols as precursors of latent electrophiles, quinone methides (QM), for lysine-targeting is demonstrated. These Michael acceptors were photogenerated for specific covalent modification of lysine residues using human serum albumin (HSA) as a model target. The reactive QM-type intermediates I or II, generated upon irradn. of 4-trifluoromethyl-1-naphthol (1)@HSA or 4-(4-trifluoromethylphenyl)phenol (2)@HSA complexes, exhibited chemoselective reactivity toward lysine residues leading to amide adducts, which was confirmed by proteomic anal. For ligand 1, the covalent modification of residues Lys106 and Lys414 (located in subdomains IA and IIIA, resp.) was obsd., whereas for ligand 2, the modification of Lys195 (in subdomain IIA) took place. Docking and mol. dynamics simulation studies provided an insight into the mol. basis of the selectivity of 1 and 2 for these HSA subdomains and the covalent modification mechanism. These studies open the opportunity of performing protein silencing by generating reactive ligands under very mild conditions (irradn.) for specific covalent modification of hidden lysine residues.398Lanfranchi, D. A.; Belorgey, D.; Muller, T.; Vezin, H.; Lanzer, M.; Davioud-Charvet, E. Exploring the trifluoromenadione core as a template to design antimalarial redox-active agents interacting with glutathione reductase. Org. Biomol. Chem. 2012, 10, 4795– 4806, DOI: 10.1039/c2ob25229e[Crossref], [PubMed], [CAS], Google Scholar398https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XnvVSrt7w%253D&md5=1f8a8106ab91264fdec3f2e1ab85b769Exploring the trifluoromenadione core as a template to design antimalarial redox-active agents interacting with glutathione reductaseLanfranchi, Don Antoine; Belorgey, Didier; Mueller, Tobias; Vezin, Herve; Lanzer, Michael; Davioud-Charvet, ElisabethOrganic & Biomolecular Chemistry (2012), 10 (24), 4795-4806CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)Menadione is the 2-methyl-1,4-naphthoquinone core used to design potent antimalarial redox-cyclers to affect the redox equil. of Plasmodium-infected red blood cells. Exploring the reactivity of fluoromethyl-1,4-naphthoquinones, in particular trifluoromenadione, under quasi-physiol. conditions in NADPH-dependent glutathione reductase reactions, is discussed in terms of chem. synthesis, electrochem., enzyme kinetics, and antimalarial activities. Multitarget-directed drug discovery is an emerging approach to the design of new antimalarial drugs. Combining in one single 1,4-naphthoquinone mol., the trifluoromenadione core with the alkyl chain at C-3 of the known antimalarial drug atovaquone, revealed a mechanism for CF3 as a leaving group. The resulting trifluoromethyl deriv. 5 showed a potent antimalarial activity per se against malarial parasites in culture.399Johann, L.; Belorgey, D.; Huang, H. H.; Day, L.; Chesse, M.; Becker, K.; Williams, D. L.; Davioud-Charvet, E. Synthesis and evaluation of 1,4-naphthoquinone ether derivatives as SmTGR inhibitors and new anti-schistosomal drugs. FEBS J. 2015, 282, 3199– 3217, DOI: 10.1111/febs.13359[Crossref], [PubMed], [CAS], Google Scholar399https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1KmurjK&md5=99d3b64dd06ee4442ea4c9a916417931Synthesis and evaluation of 1,4-naphthoquinone ether derivatives as SmTGR inhibitors and new anti-schistosomal drugsJohann, Laure; Belorgey, Didier; Huang, Hsin-Hung; Day, Latasha; Chesse, Matthieu; Becker, Katja; Williams, David L.; Davioud-Charvet, ElisabethFEBS Journal (2015), 282 (16), 3199-3217CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)New 3-benzyloxomenadiones and their difluoromethylmenadione derivs. I (R1 = Me, CHF2; R2 = H, F; R3 = COOH, CN, Cl, H, Br, OMe; R4 = CF3) were synthesized and were found to be potent and specific inhibitors of Schistosoma mansoni thioredoxin-glutathione reductase (SmTGR), which has been identified as a potential target for anti-schistosomal drugs. The compds. were also tested in enzymic assays using both human flavoenzymes, i.e. glutathione reductase (hGR) and selenium-dependent human thioredoxin reductase (hTrxR), to evaluate the specificity of the inhibition. Structure-activity relationships as well as physico- and electro-chem. studies showed a high potential for the 3-phenoxymethyl menadiones to inhibit SmTGR selectively compared to hGR and hTrxR enzymes, in particular those bearing an α-fluorophenol Me ether moiety, which improves anti-schistosomal action. Furthermore, the (substituted phenoxy)methyl menadione deriv. I (R1 = Me; R2 = H; R3 = OMe; R4 = CF3) displayed time-dependent SmTGR inactivation, correlating with unproductive NADPH-dependent redox cycling of SmTGR, and potent anti-schistosomal action in worms cultured ex vivo. In contrast, the difluoromethylmenadione analog I (R1 = CHF2; R2 = H; R3 = OMe; R4 = CF3), which inactivates SmTGR through an irreversible non-consuming NADPH-dependent process, has little killing effect in worms cultured ex vivo. Despite ex vivo activity, none of the compds. tested was active in vivo.400Eckstein, J. W.; Foster, P. G.; Finer-Moore, J.; Wataya, Y.; Santi, D. V. Mechanism-based inhibition of thymidylate synthase by 5-(trifluoromethyl)-2′-deoxyuridine 5′-monophosphate. Biochemistry 1994, 33, 15086– 15094, DOI: 10.1021/bi00254a018[ACS Full Text], [CAS], Google Scholar400https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXmvFGrt70%253D&md5=f82c290ac5cb6d16115a3517f283477aMechanism-Based Inhibition of Thymidylate Synthase by 5-(Trifluoromethyl)-2'-deoxyuridine 5'-MonophosphateEckstein, Jens W.; Foster, Paul G.; Finer-Moore, Janet; Wataya, Yusuke; Santi, Daniel V.Biochemistry (1994), 33 (50), 15086-94CODEN: BICHAW; ISSN:0006-2960.Thymidylate synthase (TS) from Lactobacillus casei is inhibited by 5-(trifluoromethyl)-2'-deoxyuridine 5'-monophosphate (CF3dUMP). CF3dUMP binds to the active site of TS in the absence of CH2H4folate, and attack of the catalytic nucleophile cysteine 198 at C6 of the pyrimidine leads to activation of the trifluoromethyl group and release of fluoride ion. Subsequently, the activated heterocycle reacts with a nucleophile of the enzyme to form a moderately stable covalent complex. Proteolytic digestion of TS treated with [2'-3H]CF3dUMP, followed by sequencing of the labeled peptides, revealed that tyrosine 146 and cysteine 198 are covalently bound to the inhibitor in the enzyme-inhibitor complex. The presence of dithiothreitol (DTT) or β-mercaptoethanol resulted in the breakdown of the covalent complex, and products from the breakdown of the complex were isolated and characterized. The three-dimensional structure of the enzyme-inhibitor complex was detd. by x-ray crystallog., clearly demonstrating covalent attachment of the nucleotide to tyrosine 146. A chem. reaction mechanism for the inhibition of TS by CF3dUMP is presented that is consistent with the kinetic, biochem., and structural results.401Begue, J.-P.; Bonnet-Delpon, D. Recent advances (1995–2005) in fluorinated pharmaceuticals based on natural products. J. Fluorine Chem. 2006, 127, 992– 1012, DOI: 10.1016/j.jfluchem.2006.05.006[Crossref], [CAS], Google Scholar401https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XnvVSru78%253D&md5=4741870ef64899a173607fecb4cf66beRecent advances (1995-2005) in fluorinated pharmaceuticals based on natural productsBegue, Jean-Pierre; Bonnet-Delpon, DanieleJournal of Fluorine Chemistry (2006), 127 (8), 992-1012CODEN: JFLCAR; ISSN:0022-1139. (Elsevier B.V.)A review. This present report is devoted to the recent advances, in these last 10 years, in fluorinated analogs of natural products developed as pharmaceuticals, marketed, registered or in clin. development. These mainly concern fluorine-substituted nucleosides, alkaloids, macrolides, steroids, amino acids and prostaglandins.402Santi, D. V.; Sakai, T. T. Thymidylate synthetase. Model studies of inhibition by 5-trifluoromethyl-2’-deoxyuridylic acid. Biochemistry 1971, 10, 3598– 3607, DOI: 10.1021/bi00795a018[ACS Full Text], [CAS], Google Scholar402https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE38XhtF2l&md5=c2006c61dbce0f15ec1a03f02f671d4eThymidylate synthetase. Model studies of inhibition by 5-trifluoromethyl-2'-deoxyuridylic acidSanti, Daniel V.; Sakai, Ted T.Biochemistry (1971), 10 (19), 3598-607CODEN: BICHAW; ISSN:0006-2960.The mechanisms of hydrolytic reactions of 5-trifluoromethyluracil and its N-alkylated derivs. provide insight into the mechanism of irreversible inhibition of thymidylate synthetase by 5-trifluoromethyl-2'-deoxyuridylic acid. All reactions appear to proceed by formation of a highly reactive intermediate having an exocyclic difluoromethylene group at the 5 position which subsequently reacts with water or hydroxide ion in a series of rapid steps to give corresponding 5-carboxyuracils. An analogous mechanism for the acylation of thymidylate synthetase by 5-trifluoromethyl-2'-deoxyuridine is proposed in which a nucleophilic group of the enzyme active-site participates in the activation of the trifluoromethyl group.403Pettersson, M.; Hou, X.; Kuhn, M.; Wager, T. T.; Kauffman, G. W.; Verhoest, P. R. Quantitative assessment of the impact of fluorine substitution on P-glycoprotein (P-gp) mediated efflux, permeability, lipophilicity, and metabolic stability. J. Med. Chem. 2016, 59, 5284– 5296, DOI: 10.1021/acs.jmedchem.6b00027[ACS Full Text], [CAS], Google Scholar403https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xos1Kjsro%253D&md5=7ca62f3c1100c6623d082d88b3113cf8Quantitative Assessment of the Impact of Fluorine Substitution on P-Glycoprotein (P-gp) Mediated Efflux, Permeability, Lipophilicity, and Metabolic StabilityPettersson, Martin; Hou, Xinjun; Kuhn, Max; Wager, Travis T.; Kauffman, Gregory W.; Verhoest, Patrick R.Journal of Medicinal Chemistry (2016), 59 (11), 5284-5296CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Strategic replacement of one or more hydrogen atoms with fluorine atom(s) is a common tactic to improve potency at a given target and/or to modulate parameters such as metabolic stability and pKa. Mol. wt. (MW) is a key parameter in design, and incorporation of fluorine is assocd. with a disproportionate increase in MW considering the van der Waals radius of fluorine vs. hydrogen. Herein we examine a large compd. data set to understand the effect of introducing fluorine on the risk of encountering P-glycoprotein mediated efflux (as measured by MDR efflux ratio), passive permeability, lipophilicity, and metabolic stability. Statistical modeling of the MDR ER data demonstrated that an increase in MW as a result of introducing fluorine atoms does not lead to higher risk of P-gp mediated efflux. Fluorine-cor. mol. wt. (MWFC), where the mol. wt. of fluorine has been subtracted, was found to be a more relevant descriptor.404Tan, E. Y.; Hartmann, G.; Chen, Q.; Pereira, A.; Bradley, S.; Doss, G.; Zhang, A. S.; Ho, J. Z.; Braun, M. P.; Dean, D. C.; Tang, W.; Kumar, S. Pharmacokinetics, metabolism, and excretion of anacetrapib, a novel inhibitor of the cholesteryl ester transfer protein, in rats and rhesus monkeys. Drug Metab. Dispos. 2010, 38, 459– 473, DOI: 10.1124/dmd.109.028696[Crossref], [PubMed], [CAS], Google Scholar404https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXjt1OrtL4%253D&md5=fe04465bd0c53e02e295698e0e038f6aPharmacokinetics, metabolism, and excretion of anacetrapib, a novel inhibitor of the cholesteryl ester transfer protein, in rats and rhesus monkeysTan, Eugene Y.; Hartmann, Georgy; Chen, Qing; Pereira, Antonio; Bradley, Scott; Doss, George; Zhang, Andy Shiqiang; Ho, Jonathan Z.; Braun, Matthew P.; Dean, Dennis C.; Tang, Wei; Kumar, SanjeevDrug Metabolism and Disposition (2010), 38 (3), 459-473CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)The pharmacokinetics and metab. of anacetrapib (MK-0859), a novel cholesteryl ester transfer protein inhibitor, were examd. in rats and rhesus monkeys. Anacetrapib exhibited a low clearance in both species and a moderate oral bioavailability of ∼38% in rats and ∼13% in monkeys. The area under the plasma concn.-time curve in both species increased in a less than dose-proportional manner over an oral dose range of 1 to 500 mg/kg. After oral administration of [14C]anacetrapib at 10 mg/kg, ∼80 and 90% of the radioactive dose was recovered over 48 h postdose from rats and monkeys, resp. The majority of the administered radioactive dose was excreted unchanged in feces in both species. Biliary excretion of radioactivity accounted for ∼15% and urinary excretion for less than 2% of the dose. Thirteen metabolites, resulting from oxidative and secondary glucuronic acid conjugation, were identified in rat and monkey bile. The main metabolic pathways consisted of O-demethylation (M1) and hydroxylation on the biphenyl moiety (M2) and hydroxylation on the iso-Pr side chain (M3); these hydroxylations were followed by O-glucuronidation of these metabolites. A glutathione adduct (M9), an olefin metabolite (M10), and a propionic acid metabolite (M11) also were identified. In addn. to parent anacetrapib, M1, M2, and M3 metabolites were detected in rat but not in monkey plasma. Overall, it appears that anacetrapib exhibits a low-to-moderate degree of absorption after oral dosing and majority of the absorbed dose is eliminated via oxidn. to a series of hydroxylated metabolites that undergo conjugation with glucuronic acid before excretion into bile.405Bowman, L.; Hopewell, J. C.; Chen, F.; Wallendszus, K.; Stevens, W.; Collins, R.; Wiviott, S. D.; Cannon, C. P.; Braunwald, E.; Sammons, E.; Landray, M. J. Effects of anacetrapib in patients with atherosclerotic vascular disease. N. Engl. J. Med. 2017, 377, 1217– 1227, DOI: 10.1056/NEJMoa1706444[Crossref], [PubMed], [CAS], Google Scholar405https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1cbit1Gmsg%253D%253D&md5=8f152d6a2e2d83df68f4b511bf59164bEffects of Anacetrapib in Patients with Atherosclerotic Vascular DiseaseBowman Louise; Hopewell Jemma C; Chen Fang; Wallendszus Karl; Stevens William; Collins Rory; Wiviott Stephen D; Cannon Christopher P; Braunwald Eugene; Wiviott Stephen D; Cannon Christopher P; Braunwald Eugene; Sammons Emily; Landray Martin JThe New England journal of medicine (2017), 377 (13), 1217-1227 ISSN:.BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .).406Gotto, A. M., Jr.; Cannon, C. P.; Li, X. S.; Vaidya, S.; Kher, U.; Brinton, E. A.; Davidson, M.; Moon, J. E.; Shah, S.; Dansky, H. M.; Mitchel, Y.; Barter, P. Evaluation of lipids, drug concentration, and safety parameters following cessation of treatment with the cholesteryl ester transfer protein inhibitor anacetrapib in patients with or at high risk for coronary heart disease. Am. J. Cardiol. 2014, 113, 76– 83, DOI: 10.1016/j.amjcard.2013.08.041[Crossref], [PubMed], [CAS], Google Scholar406https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhslajsLzN&md5=fa70e20a5a463a78c9df31c4bf66f588Evaluation of Lipids, Drug Concentration, and Safety Parameters Following Cessation of Treatment With the Cholesteryl Ester Transfer Protein Inhibitor Anacetrapib in Patients With or at High Risk for Coronary Heart DiseaseGotto, Antonio M.; Cannon, Christopher P.; Li, Xiujiang Susie; Vaidya, Sanskruti; Kher, Uma; Brinton, Eliot A.; Davidson, Michael; Moon, Jennifer E.; Shah, Sukrut; Dansky, Hayes M.; Mitchel, Yale; Barter, PhilipAmerican Journal of Cardiology (2014), 113 (1), 76-83CODEN: AJCDAG; ISSN:0002-9149. (Elsevier)The aim of this study was to assess the effects on lipids and safety during a 12-wk reversal period after 18 mo of treatment with anacetrapib. The cholesteryl ester transfer protein inhibitor anacetrapib was previously shown to reduce low-d. lipoprotein cholesterol by 39.8% (estd. using the Friedewald equation) and increase high-d. lipoprotein (HDL) cholesterol by 138.1%, with an acceptable side-effect profile, in patients with or at high risk for coronary heart disease in the Detg. the Efficacy and Tolerability of CETP Inhibition With Anacetrapib (DEFINE) trial. A total of 1,398 patients entered the 12-wk reversal-phase study, either after completion of the active-treatment phase or after early discontinuation of the study medication. In patients allocated to anacetrapib, placebo-adjusted mean percentage decreases from baseline were obsd. at 12 wk off the study drug for Friedewald-calcd. low-d. lipoprotein cholesterol (18.6%), non-HDL cholesterol (17.6%), and apolipoprotein B (10.2%); placebo-adjusted mean percentage increases were obsd. for HDL cholesterol (73.0%) and apolipoprotein A-I (24.5%). Residual plasma anacetrapib levels (about 40% of on-treatment apparent steady-state trough levels) were also detected 12 wk after cessation of anacetrapib. No clin. important elevations in liver enzymes, blood pressure, electrolytes, or adverse experiences were obsd. during the reversal phase. Preliminary data from a small cohort (n = 30) revealed the presence of low concns. of anacetrapib in plasma 2.5 to 4 years after the last anacetrapib dose. In conclusion, after the cessation of active treatment, anacetrapib plasma lipid changes and drug levels decreased to approx. 40% of on-treatment trough levels at 12 wk after dosing, but modest HDL cholesterol elevations and low drug concns. were still detectable 2 to 4 years after the last dosing.407Johns, D. G.; LeVoci, L.; Krsmanovic, M.; Lu, M.; Hartmann, G.; Xu, S.; Wang, S. P.; Chen, Y.; Bateman, T.; Blaustein, R. O. Characterization of anacetrapib distribution into the lipid droplet of adipose tissue in mice and human cultured adipocytes. Drug Metab. Dispos. 2019, 47, 227– 233, DOI: 10.1124/dmd.118.084525[Crossref], [PubMed], [CAS], Google Scholar407https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXotlKiu7k%253D&md5=b8437c0827e69d455fa217148e288fc6Characterization of anacetrapib distribution into the lipid droplet of adipose tissue in mice and human cultured adipocytesJohns, Douglas G.; Levoci, Lauretta; Krsmanovic, Mihajlo; Lu, Min; Hartmann, Georgy; Xu, Suoyu; Wang, Sheng-Ping; Chen, Ying; Bateman, Thomas; Blaustein, Robert O.Drug Metabolism & Disposition (2019), 47 (3), 227-233CODEN: DMDSAI; ISSN:1521-009X. (American Society for Pharmacology and Experimental Therapeutics)Anacetrapib is an inhibitor of cholesteryl ester transfer protein (CETP), assocd. with redn. in LDL cholesterol and increase in HDL cholesterol in hypercholesterolemic patients. Anacetrapib was not taken forward into filing/registration as a newdrug for coronary artery diease, despite the observation of a ∼9% redn. in cardiovascular risk in a large phase III cardiovascular outcomes trial (REVEAL). Anacetrapib displayed no adverse effects throughout extensive preclin. safety evaluation, and no major safety signals were obsd. in clin. trials studying anacetrapib, including REVEAL. However, anacetrapib demonstrated a long terminal half-life in all species, thought to be due, in part, to distribution into adipose tissue. We sought to understand the dependence of anacetrapib's long half-life on adipose tissue and to explore potential mechanisms that might contribute to the phenomenon. In mice, anacetrapib localized primarily to the lipid droplet of adipocytes in white adipose tissue; in vitro, anacetrapib entry into cultured human adipocytes depended on the presence of a mature adipocyte and lipid droplet but did not require active transport. In vivo, the entry of anacetrapib into adipose tissue did not require lipase activity, as the distribution of anacetrapib into adipose was-not affected by systemic lipase inhibition using poloaxamer-407, a systemic lipase inhibitor. The data from these studies support the notion that the entry of anacetrapib into adipose tissue/lipid droplets does not require active transport, nor does it require mobilization or entry of fat into adipose via lipolysis.408Hartmann, G.; Kumar, S.; Johns, D.; Gheyas, F.; Gutstein, D.; Shen, X.; Burton, A.; Lederman, H.; Lutz, R.; Jackson, T.; Chavez-Eng, C.; Mitra, K. Disposition into adipose tissue determines accumulation and elimination kinetics of the cholesteryl ester transfer protein inhibitor anacetrapib in mice. Drug Metab. Dispos. 2016, 44, 428– 434, DOI: 10.1124/dmd.115.067736[Crossref], [PubMed], [CAS], Google Scholar408https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28rlsVSlsw%253D%253D&md5=889e0108f486863be71af1807c6e4f40Disposition into Adipose Tissue Determines Accumulation and Elimination Kinetics of the Cholesteryl Ester Transfer Protein Inhibitor Anacetrapib in MiceHartmann Georgy; Kumar Sanjeev; Johns Douglas; Gheyas Ferdous; Gutstein David; Shen Xiaolan; Burton Aimee; Lederman Harmony; Lutz Ryan; Jackson Tonya; Chavez-Eng Cynthia; Mitra KaushikDrug metabolism and disposition: the biological fate of chemicals (2016), 44 (3), 428-34 ISSN:.The cholesteryl ester transfer protein (CETP) inhibitor anacetrapib exhibits a long terminal half-life (t1/2) in humans; however, the dispositional mechanisms that lead to this long t1/2 are still being elucidated. As it is hypothesized that disposition into adipose tissue and binding to CETP might play a role, we sought to delineate the relative importance of these factors using a preclinical animal model. A multiple-dose pharmacokinetic study was conducted in C57BL6 wild-type (WT) lean, WT diet-induced obese (DIO), natural flanking region (NFR) CETP-transgenic lean, and NFR-DIO mice. Mice were dosed orally with 10 mg/kg anacetrapib daily for 42 days. Drug concentrations in blood, brown and white adipose tissue, liver, and brain were measured up to 35 weeks postdose. During dosing, a 3- to 9-fold accumulation in 72-hour postdose blood concentrations of anacetrapib was observed. Drug concentrations in white adipose tissue accumulated ∼20- to 40-fold, whereas 10- to 17-fold accumulation occurred in brown adipose and approximately 4-fold in liver. Brain levels were very low (<0.1 μM), and a trend of accumulation was not seen. The presence of CETP as well as adiposity seems to play a role in determining the blood concentrations of anacetrapib. The highest blood concentrations were observed in NFR DIO mice, whereas the lowest concentrations were seen in WT lean mice. In adipose and liver tissue, higher concentrations were seen in DIO mice, irrespective of the presence of CETP. This finding suggests that white adipose tissue serves as a potential depot and that disposition into adipose tissue governs the long-term kinetics of anacetrapib in vivo.409Giudicelli, J. F.; Richer, C.; Berdeaux, A. Preliminary assessment of flutiorex, a new anorectic drug, in man. Br. J. Clin. Pharmacol. 1976, 3, 113– 121, DOI: 10.1111/j.1365-2125.1976.tb00578.x[Crossref], [PubMed], [CAS], Google Scholar409https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE28XkvVOntbg%253D&md5=a6a920a7186d45a423130e8d549c2f26Preliminary assessment of flutiorex, a new anorectic drug, in manGiudicelli, J. F.; Richer, Christine; Berdeaux, A.British Journal of Clinical Pharmacology (1976), 3 (1), 113-21CODEN: BCPHBM; ISSN:0306-5251.Flutiorex-HCl (I-HCl) [59960-78-0] exerted a significant anorectic effect and was approx. twice as potent as fenfluramine-HCl [404-82-0] in healthy volunteers. I induces a definite α-adrenergic sympathomimetic stimulation as was shown by a rise in systolic blood pressure and the development of marked mydriasis. I was a central nervous system stimulant producing an increase in crit. flicker frequency. It did not, however, influence psychomotor coordination as reflected in the pursuit rotor test. Serial detns. of blood and urine levels of I and its deethylated metabolite, norflutiorex [52771-23-0], showed that I was rapidly absorbed and deethylated, accumulated in large quantities in the tissues and, like its metabolite, was excreted in the urine in very small quantities. Blood levels of norflutiorex appear to remain elevated longer than those of I.410Silverstone, T.; Fincham, J.; Plumley, J. An evaluation of the anorectic activity in man of a sustained release formulation of tiflorex. Br. J. Clin. Pharmacol. 1979, 7, 353– 356, DOI: 10.1111/j.1365-2125.1979.tb00945.x[Crossref], [PubMed], [CAS], Google Scholar410https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1MXktlaqu7Y%253D&md5=51cfe71c0cc5b2e6fd345334337b775cAn evaluation of the anorectic activity in man of a sustained release formulation of tiflorexSilverstone, T.; Fincham, Jill; Plumley, JillBritish Journal of Clinical Pharmacology (1979), 7 (4), 353-6CODEN: BCPHBM; ISSN:0306-5251.In healthy female volunteers, a redn. in hunger following a slow release form of tiflorex (I) [53993-67-2] (20 mg, orally) was obsd. some 5-7 h after administration; no significant redn. in hunger occurred after placebo. A significant redn. in food intake occurred at a time corresponding to, and slightly later than, the subjective anorectic effect. I produced no effect on pulse rate or blood pressure, but it did induce some mydriasis. There was no evidence of CNS stimulation after I, and the drug had no influence on flicker fusion frequency or psychomotor performance. A redn. in subjective arousal was obsd. after 3 h, but I caused no other significant change in subjective measures of arousal or mood. Only slight sleep disturbance was reported; subjective reports of headache were more frequent after drug administration than after placebo, occurring in 7 out of 12 active drug exposures. Side effects were noted on 11 occasions after I, compared with 5 occasions after placebo.411Benoit, E.; Cresteil, T.; Riviere, J. L.; Delatour, P. Specific and enantioselective sulfoxidation of an aryl-trifluoromethyl sulfide by rat liver cytochromes P-450. Drug Metab. Dispos. 1992, 20, 877– 881[PubMed], [CAS], Google Scholar411https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXhtFKit7o%253D&md5=4f83b94031598759d705833a9b3514c4Specific and enantioselective sulfoxidation of an aryl-trifluoromethyl sulfide by rat liver cytochromes P-450Benoit, Etienne; Cresteil, Thierry; Riviere, Jean Louis; Delatour, PaulDrug Metabolism and Disposition (1992), 20 (6), 877-81CODEN: DMDSAI; ISSN:0090-9556.Evidence based on thermal stability and enzyme inhibition data suggests that the sulfoxidn. of the drug toltrazuril by rat liver microsomes is catalyzed by different cytochromes P 450. Pretreatment of rats by different inducers (phenobarbital, 3-methylcholanthrene, dexamethasone, and triacetyloleanodomycin) results in a 2.1-, 2.6-, 2.9-, and 1.8-fold increase, resp., in the rate of sulfoxidn. The highest increase (8.4-fold) was obsd. after treatment of microsomes from triacetyloleandomycin-treated animals by potassium ferricyanide. Castration and aging also modify the sulfoxidase activity. The relative rate of formation of the two toltrazuril enantiomers [(A)- and (B)-sulfoxides] depends on the source of the microsomes, suggesting that different cytochromes P 450 have different stereoselectivities.412Benoit, E.; Buronfosse, T.; Delatour, P. Effect of cytochrome P-450 1A induction on enantioselective metabolism and pharmacokinetics of an aryltrifluoromethyl sulfide in the rat. Chirality 1994, 6, 372– 377, DOI: 10.1002/chir.530060503[Crossref], [PubMed], [CAS], Google Scholar412https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXjs1GksA%253D%253D&md5=87b0af36f97233f6e2490d52f70bd250Effect of cytochrome P-450 1A induction on enantioselective metabolism and pharmacokinetics of an aryltrifluoromethyl sulfide in the ratBenoit, Etienne; Buronfosse, Thierry; Delatour, PaulChirality (1994), 6 (5), 372-7CODEN: CHRLEP; ISSN:0899-0042.The pharmacokinetics of the antiparasitic drug toltrazuril (1-methyl-3-[3-methyl-4-[4-[trifluoromethyl]thio]phenoxy]phenyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione) were studied in the rat following pretreatment with 3-methylcholanthrene, an inducer of rat liver cytochrome P 450 1A. The induction markedly modified the pharmacokinetics of the compd., leading to a decrease in the AUC value for toltrazuril sulfoxide. The results were explained on the basis of previous results from the authors lab. relating to the product enantioselectivity of the formation of the sulfoxide and the substrate enantioselectivity of the subsequent formation of the sulfone.413Mas-Chamberlin, C.; Gillet, G.; André, J.; Gomeni, R.; Dring, L. G.; Morselli, P. L. The metabolism and kinetics of tiflorex in the rat. Drug Metab. Dispos. 1981, 9, 150– 155[PubMed], [CAS], Google Scholar413https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXktVSitbo%253D&md5=24341578b2f862cfa323177c402f5cd2The metabolism and kinetics of tiflorex in the ratMas-Chamberlin, Claire; Gillet, Gerard; Andre, Joelle; Gomeni, Roberto; Dring, L. Graham; Morselli, Paolo L.Drug Metabolism and Disposition (1981), 9 (2), 150-5CODEN: DMDSAI; ISSN:0090-9556.14C-labeled tiflorex-HCl (I) [59960-78-0] given either orally or i.v. to male rats (10 mg/kg; 250 μCi) was well absorbed orally; >70% of the dose was excreted in the urine in the 1st 48 h after dosing by either route of administration. Part of the dose (10%) was excreted in the feces after i.v. administration; biliary excretion may be a route of elimination. The major route of metab. was S-oxidn. to give the sulfoxides and sulfones of tiflorex (7% each) and nortiflorex [52771-24-1] which were excreted together with the unchanged drug (1%) in the 0- to 48-h urine. Drug was rapidly absorbed orally, max. levels was attained within 30 min. The plasma half-life for the elimination phase of the unchanged drug was relatively long (7.5 h) compared with the metabolites (2.5 h) with the exception of nortiflorex sulfone [82560-96-1] (9.8 h) and 2 as yet unidentified metabolites which had half-lives in excess of 24 h. The latter 3 compds. were responsible for the relatively long plasma half-life of total radioactivity (∼13 h). The ratio of the areas under the plasma curve for unchanged drug indicated a low bioavailability (30%). Apparently, the predominant route of metab. of this group of compds. in the rat, p-hydroxylation, was blocked by the trifluoromethylthio group, with consequent emphasis on S-oxidn.414Karadzovska, D.; Seewald, W.; Browning, A.; Smal, M.; Bouvier, J.; Giraudel, J. M. Pharmacokinetics of monepantel and its sulfone metabolite, monepantel sulfone, after intravenous and oral administration in sheep. J. Vet. Pharmacol. Ther. 2009, 32, 359– 367, DOI: 10.1111/j.1365-2885.2008.01052.x[Crossref], [PubMed], [CAS], Google Scholar414https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtValtbjM&md5=96efb9456a5d61f4b1321ecd7ad08e0aPharmacokinetics of monepantel and its sulfone metabolite, monepantel sulfone, after intravenous and oral administration in sheepKaradzovska, D.; Seewald, W.; Browning, A.; Smal, M.; Bouvier, J.; Giraudel, J. M.Journal of Veterinary Pharmacology and Therapeutics (2009), 32 (4), 359-367CODEN: JVPTD9; ISSN:0140-7783. (Wiley-Blackwell)The pharmacokinetic properties of the developmental Amino-Acetonitrile Deriv. (AAD), monepantel and its sulfone metabolite, monepantel sulfone were investigated in sheep following i.v. and oral administrations. The sulfone metabolite was rapidly formed and predominated over monepantel 4 h after dosing, irresp. of the route of administration. The steady-state vol. of distribution, total body clearance and mean residence time of monepantel were 7.4 L/kg, 1.49 L/(kg/h) and 4.9 h, resp. and 31.2 L/kg, 0.28 L/(kg/h) and 111 h, resp. for monepantel sulfone. The overall bioavailability of monepantel was 31%, but it was demonstrated that approx. the same amt. of monepantel sulfone was produced whether monepantel was given i.v. or orally (AUC(0-∞) oral/AUC(0-∞) i.v. of 94% for monepantel sulfone), making oral administration a very efficient route of administration for monepantel in terms of the amt. of sulfone metabolite generated. Because monepantel sulfone is the main chem. entity present in sheep blood after monepantel administration and because it is also an active metabolite, its pharmacokinetic properties are of primary importance for the interpretation of future residue and efficacy studies. Overall, these pharmacokinetic data aid in the evaluation of monepantel as an oral anthelmintic in sheep.415Lim, J. H.; Kim, M. S.; Hwang, Y. H.; Song, I. B.; Park, B. K.; Yun, H. I. Pharmacokinetics of toltrazuril and its metabolites, toltrazuril sulfoxide and toltrazuril sulfone, after a single oral administration to pigs. J. Vet. Med. Sci. 2010, 72, 1085– 1087, DOI: 10.1292/jvms.09-0524[Crossref], [PubMed], [CAS], Google Scholar415https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtFKrtLrE&md5=cfca7a5e60162cfa3c525d5cb3b05980Pharmacokinetics of toltrazuril and its metabolites, toltrazuril sulfoxide and toltrazuril sulfone, after a single oral administration to pigsLim, Jong-Hwan; Kim, Myoung-Seok; Hwang, Youn-Hwan; Song, In-Bae; Park, Byung-Kwon; Yun, Hyo-InJournal of Veterinary Medical Science (2010), 72 (8), 1085-1087CODEN: JVMSEQ; ISSN:0916-7250. (Japanese Society of Veterinary Science)Toltrazuril (TZR) is a triazine-based antiprotozoal agent. Following a single oral administration of TZR at 10 and 20 mg/kg to male pigs, the mean TZR concn. in plasma peaked at 4.24 and 8.18 μg/mL at 15.0 and 12.0 h post-dose, resp. TZR absorbed was rapidly converted to the short-lived intermediary metabolite toltrazuril sulfoxide (TZR-SO), and then metabolized to the reactive toltrazuril sulfone (TZR-SO2). TZR-SO2 was actually more slowly eliminated, with av. half-lives of 231 and 245 h, compared with TZR (48.7 and 68.9 h) or TZR-SO (51.9 and 53.2 h) in the 10 and 20 mg/kg groups, resp. This study demonstrates that TZR metabolizes to TZR-SO2 having a long-terminal half-life, enabling the persistent clin. efficacy in the treatment of I. suis infection. In contrast, special consideration should be given to the residual of TZR-SO2.416Ghiazza, C.; Billard, T.; Dickson, C.; Tlili, A.; Gampe, C. M. Chalcogen OCF3 isosteres modulate drug properties without introducing inherent liabilities. ChemMedChem 2019, 14, 1586– 1589, DOI: 10.1002/cmdc.201900452[Crossref], [PubMed], [CAS], Google Scholar416https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1WisrjM&md5=d711d2096dfa2ed308a365e03e67e52cChalcogen OCF3 Isosteres Modulate Drug Properties without Introducing Inherent LiabilitiesGhiazza, Clement; Billard, Thierry; Dickson, Callum; Tlili, Anis; Gampe, Christian M.ChemMedChem (2019), 14 (17), 1586-1589CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)The synthesis of SCF3 as well as SeCF3 isosteres of two OCF3-contg. drugs was achieved through visible light and copper-catalyzed processes. Herein, we show that chalcogen replacement modulates physicochem. and ADME properties without introducing intrinsic liabilities. The SCF3 and SeCF3 groups are more lipophilic than their oxygen counterpart; however, microsomal stability is unchanged, indicating that these mol. changes may be beneficial for in vivo half-life. Enabled by modern synthetic methods, we present the chalcogen-CF3 groups as potential key players for future fluorinated pharmaceuticals.417Yu, L.; Ternansky, R. J.; Victoria, E. J.; Chang, J.; Coutts, S. M. The structure-activity relationships of a series of suicide inhibitors of phospholipase A2. Bioorg. Med. Chem. Lett. 1998, 8, 2129– 2132, DOI: 10.1016/S0960-894X(98)00378-3[Crossref], [PubMed], [CAS], Google Scholar417https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXmtVKrur4%253D&md5=b83cf378d87ef0123bec65340f02787fThe structure-activity relationships of a series of suicide inhibitors of phospholipase A2Yu, Lin; Ternansky, Robert J.; Victoria, Edward J.; Chang, Julia; Coutts, Stephen M.Bioorganic & Medicinal Chemistry Letters (1998), 8 (16), 2129-2132CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science Ltd.)A series of mechanism-based inhibitors of phospholipase A2 (SIBLINKS) were synthesized. These new SIBLINKS are phospholipid analogs that contain a para-substituted Ph 3,3-dimethylglutaryl group in the place of the sn-2 acyl chain. The effect of the Ph leaving group on inhibitory activity was studied by varying the electron-withdrawing ability of the para-substituted group. A strong correlation was obsd. between the leaving group potential of the suicide inhibitor and the inhibitory activity of the deriv. toward cobra venom phospholipase A2.418Pearce, R. E.; Leeder, J. S.; Kearns, G. L. Biotransformation of fluticasone: in vitro characterization. Drug Metab. Dispos. 2006, 34, 1035– 1040, DOI: 10.1124/dmd.105.009043[Crossref], [PubMed], [CAS], Google Scholar418https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XlsVOnuro%253D&md5=f6a3f266bf4d82a2039fb3bff1fdc69bBiotransformation of Fluticasone: in vitro characterizationPearce, Robin E.; Leeder, J. Steven; Kearns, Gregory L.Drug Metabolism and Disposition (2006), 34 (6), 1035-1040CODEN: DMDSAI; ISSN:0090-9556. (American Society for Pharmacology and Experimental Therapeutics)Fluticasone propionate (FTP) is a synthetic trifluorinated glucocorticoid with potent anti-inflammatory action that is commonly used in patients with asthma. After oral or intranasal administration, FTP undergoes rapid hepatic biotransformation; the principal metabolite formed is a 17β-carboxylic acid deriv. (M1). M1 formation has been attributed largely to cytochrome P 450 3A4 (CYP3A4); however, there are no published data that confirm this assertion. Hence, in vitro studies were conducted to det. the role that human P450s play in the metab. of FTP. Consistent with in vivo data, human liver microsomes catalyzed the formation of a single metabolite (M1) at substrate concns. ≤10 μM (mean plasma Cmax = 1 nM). Under these conditions, the kinetics of M1 formation in human liver microsomes were consistent with those of a single enzyme (Km ≃ 5 μM). Formation of M1 correlated significantly (r > 0.95) with CYP3A4/5 activities in a panel of human liver microsomes (n = 14) and was markedly impaired by the CYP3A inhibitor ketoconazole (>94%) but not by inhibitors of other P 450 enzymes (≤10%). Studies with a panel of cDNA-expressed enzymes revealed that M1 formation was catalyzed primarily by CYP3A enzymes at FTP concns. ≤1 μM. M1 formation was catalyzed by P450s 3A4, 3A5, and 3A7; in vitro intrinsic clearance values (Vmax/Km) were comparable for all three CYP3A enzymes. These results suggest that at pharmacol. relevant concns., biotransformation of FTP to M1 is mediated predominantly by CYP3A enzymes in the liver.419Westphal, M. V.; Wolfstadter, B. T.; Plancher, J. M.; Gatfield, J.; Carreira, E. M. Evaluation of tert-butyl isosteres: case studies of physicochemical and pharmacokinetic properties, efficacies, and activities. ChemMedChem 2015, 10, 461– 469, DOI: 10.1002/cmdc.201402502[Crossref], [PubMed], [CAS], Google Scholar419https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtlWis7w%253D&md5=06f976396f0ec1bdb7a7f93fdd3808f3Evaluation of tert-Butyl isosteres: case studies of physicochemical and pharmacokinetic properties, efficacies, and activitiesWestphal, Matthias V.; Wolfstaedter, Bernd T.; Plancher, Jean-Marc; Gatfield, John; Carreira, Erick M.ChemMedChem (2015), 10 (3), 461-469CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)The tert-Bu group is a common motif in medicinal chem. Its incorporation into bioactive compds. is often accompanied by unwanted property modulation, such as increased lipophilicity and decreased metabolic stability. Several alternative substituents are available for the drug discovery process. Herein, physicochem. data of two series of drug analogs of bosentan and vercirnon are documented as part of a comparative study of tert-Bu, pentafluorosulfanyl, trifluoromethyl, bicyclo[1.1.1]pentyl, and cyclopropyl-trifluoromethyl substituents.420Sowaileh, M. F.; Hazlitt, R. A.; Colby, D. A. Application of the pentafluorosulfanyl group as a bioisosteric replacement. ChemMedChem 2017, 12, 1481– 1490, DOI: 10.1002/cmdc.201700356[Crossref], [PubMed], [CAS], Google Scholar420https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsV2ju7fK&md5=d7ebb1fd3f7f0f380f16608a16ffefc6Application of the Pentafluorosulfanyl Group as a Bioisosteric ReplacementSowaileh, Munia F.; Hazlitt, Robert A.; Colby, David A.ChemMedChem (2017), 12 (18), 1481-1490CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)The success of fluorinated mols. in drug design has led medicinal chemists to search for new fluorine-contg. substituents. A major recently developed group is the pentafluorosulfanyl group. This group is stable under physiol. conditions and displays unique phys. and chem. properties. There are currently few synthetic methods to install the SF5 group, yet efforts to integrate this group into lead optimization continue unabated. Typically, the SF5 group has been used as a replacement for trifluoromethyl, tert-Bu, halogen, or nitro groups. In this review, the use of the SF5 group as a bioisosteric replacement for each of these three functionalities is compared and contrasted across various groups of biol. active mols. The organization and presentation of these data should be instructive to medicinal chemists considering to design synthetic strategies to access SF5-substituted mols.421Hansch, C.; Muir, R. M.; Fujita, T.; Maloney, P. P.; Geiger, F.; Streich, M. The correlation of biological activity of plant growth regulators and chloromycetin derivatives with Hammett constants and partition coefficients. J. Am. Chem. Soc. 1963, 85, 2817– 2824, DOI: 10.1021/ja00901a033[ACS Full Text], [CAS], Google Scholar421https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF3sXksFKhtbk%253D&md5=ce902ea696b325cb22b74d58c9fcdd6cThe correlation of biological activity of plant growth regulators and chloromycetin derivatives with Hammett constants and partition coefficientsHansch, Corwin; Muir, Robert M.; Fujita, Toshio; Maloney, Peyton P.; Geiger, Fred; Streich, MargaretJournal of the American Chemical Society (1963), 85 (18), 2817-24CODEN: JACSAT; ISSN:0002-7863.An equation using 2 exptl. based variables, σ and π, was developed for correlating the effect of a given substituent on the biological activity of a parent compd.; is the Hammett substituent const. and π is an analogous const. representing the difference in the logarithms of the partition coeffs. of the substituted and unsubstituted compds. (π = log Px - log PH). The value of this equation was tested on 2 systems of biol. active mols.: the phenoxyacetic acids and chloromycetin analogs. By using π and a it becomes possible to disentangle three of the most important parameters governing the biol. activity of org. compns.: steric, electronic, and rate of penetration.422Welch, J. T.; Lim, D. S. The synthesis and biological activity of pentafluorosulfanyl analogs of fluoxetine, fenfluramine, and norfenfluramine. Bioorg. Med. Chem. 2007, 15, 6659– 6666, DOI: 10.1016/j.bmc.2007.08.012[Crossref], [PubMed], [CAS], Google Scholar422https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVCisbfP&md5=5aa869f37f1c306c1879cbb75aa251fbThe synthesis and biological activity of pentafluorosulfanyl analogs of fluoxetine, fenfluramine, and norfenfluramineWelch, John T.; Lim, Dong SungBioorganic & Medicinal Chemistry (2007), 15 (21), 6659-6666CODEN: BMECEP; ISSN:0968-0896. (Elsevier Ltd.)The trifluoromethyl group of fluoxetine, fenfluramine, and norfenfluramine was substituted by the pentafluorosulfanyl group. On examn. of the efficacy of the pentafluorosulfanyl contg. compds. as inhibitors of 5-hydroxytryptamine receptors, it was found that substitution could lead to enhanced selectivity and in the case of the pentafluorosulfanyl analog of fenfluramine (I) it significantly enhanced potency against the 5-HT2b, 5-HT2c, and 5-HT6 receptors.423Wipf, P.; Mo, T.; Geib, S. J.; Caridha, D.; Dow, G. S.; Gerena, L.; Roncal, N.; Milner, E. E. Synthesis and biological evaluation of the first pentafluorosulfanyl analogs of mefloquine. Org. Biomol. Chem. 2009, 7, 4163– 4165, DOI: 10.1039/b911483a[Crossref], [PubMed], [CAS], Google Scholar423https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtFyqtLzL&md5=4bc80514b8db8ff1714d76ee2ce6a631Synthesis and biological evaluation of the first pentafluorosulfanyl analogs of mefloquineWipf, Peter; Mo, Tingting; Geib, Steven J.; Caridha, Diana; Dow, Geoffrey S.; Gerena, Lucia; Roncal, Norma; Milner, Erin E.Organic & Biomolecular Chemistry (2009), 7 (20), 4163-4165CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)Two novel SF5 analogs of the antimalarial agent mefloquine were synthesized in 5 steps and 10-23% overall yields and found to have improved activity and selectivity against malaria parasites.424Hendriks, C. M.; Penning, T. M.; Zang, T.; Wiemuth, D.; Grunder, S.; Sanhueza, I. A.; Schoenebeck, F.; Bolm, C. Pentafluorosulfanyl-containing flufenamic acid analogs: Syntheses, properties and biological activities. Bioorg. Med. Chem. Lett. 2015, 25, 4437– 4440, DOI: 10.1016/j.bmcl.2015.09.012[Crossref], [PubMed], [CAS], Google Scholar424https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsV2nt7fK&md5=101cf19c1d00ef749a1c36b5ff6ab091Pentafluorosulfanyl-containing flufenamic acid analogs: Syntheses, properties and biological activitiesHendriks, Christine M. M.; Penning, Trevor M.; Zang, Tianzhu; Wiemuth, Dominik; Gruender, Stefan; Sanhueza, Italo A.; Schoenebeck, Franziska; Bolm, CarstenBioorganic & Medicinal Chemistry Letters (2015), 25 (20), 4437-4440CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Pentafluorosulfanyl-contg. analogs of flufenamic acid have been synthesized in high yields. Computationally, pKa, Log P and Log D values have been detd. Initial bioactivity studies reveal effects as ion channel modulators and inhibitory activities on aldo-keto reductase 1C3 (AKR1C3) as well as COX-1 and COX-2.425Zhang, Y.; Wang, Y.; He, C.; Liu, X.; Lu, Y.; Chen, T.; Pan, Q.; Xiong, J.; She, M.; Tu, Z.; Qin, X.; Li, M.; Tortorella, M. D.; Talley, J. J. Pentafluorosulfanyl-substituted benzopyran analogues as new cyclooxygenase-2 inhibitors with excellent pharmacokinetics and efficacy in blocking inflammation. J. Med. Chem. 2017, 60, 4135– 4146, DOI: 10.1021/acs.jmedchem.6b01484[ACS Full Text], [CAS], Google Scholar425https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXntVyru7Y%253D&md5=9a7a5a33f913ad2981dc14f57929cda0Pentafluorosulfanyl-Substituted Benzopyran Analogues As New Cyclooxygenase-2 Inhibitors with Excellent Pharmacokinetics and Efficacy in Blocking InflammationZhang, Yanmei; Wang, Yican; He, Chuang; Liu, Xiaorong; Lu, Yongzhi; Chen, Tingting; Pan, Qiong; Xiong, Jingfang; She, Miaoqin; Tu, Zhengchao; Qin, Xiaochu; Li, Minke; Tortorella, Micky D.; Talley, John J.Journal of Medicinal Chemistry (2017), 60 (10), 4135-4146CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)In this report, we disclose the design and synthesis of a series of pentafluorosulfanyl (SF5) benzopyran derivs. as novel COX-2 inhibitors with improved pharmacokinetic and pharmacodynamic properties. The pentafluorosulfanyl compds. showed both potency and selectivity for COX-2 and demonstrated efficacy in several murine models of inflammation and pain. More interestingly, one of the compds., I, revealed exceptional efficacy in the adjuvant induced arthritis (AIA) model, achieving an ED50 as low as 0.094 mg/kg. In addn., the pharmacokinetics of compd. I in rat revealed a half-life in excess of 12 h and plasma drug concns. well above its IC90 for up to 40 h. When I was dosed just two times a week in the AIA model, efficacy was still maintained. Overall, drug I and other analogs are suitable candidates that merit further investigation for the treatment of inflammation and pain as well as other diseases where COX-2 and PGE2 play a role in their etiol.426Phillips, M. A.; Lotharius, J.; Marsh, K.; White, J.; Dayan, A.; White, K. L.; Njoroge, J. W.; El Mazouni, F.; Lao, Y.; Kokkonda, S.; Tomchick, D. R.; Deng, X.; Laird, T.; Bhatia, S. N.; March, S.; Ng, C. L.; Fidock, D. A.; Wittlin, S.; Lafuente-Monasterio, M.; Benito, F. J.; Alonso, L. M.; Martinez, M. S.; Jimenez-Diaz, M. B.; Bazaga, S. F.; Angulo-Barturen, I.; Haselden, J. N.; Louttit, J.; Cui, Y.; Sridhar, A.; Zeeman, A. M.; Kocken, C.; Sauerwein, R.; Dechering, K.; Avery, V. M.; Duffy, S.; Delves, M.; Sinden, R.; Ruecker, A.; Wickham, K. S.; Rochford, R.; Gahagen, J.; Iyer, L.; Riccio, E.; Mirsalis, J.; Bathhurst, I.; Rueckle, T.; Ding, X.; Campo, B.; Leroy, D.; Rogers, M. J.; Rathod, P. K.; Burrows, J. N.; Charman, S. A. A long-duration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria. Sci. Transl. Med. 2015, 7, 296ra111, DOI: 10.1126/scitranslmed.aaa6645427McCarthy, J. S.; Lotharius, J.; Ruckle, T.; Chalon, S.; Phillips, M. A.; Elliott, S.; Sekuloski, S.; Griffin, P.; Ng, C. L.; Fidock, D. A.; Marquart, L.; Williams, N. S.; Gobeau, N.; Bebrevska, L.; Rosario, M.; Marsh, K.; Mohrle, J. J. Safety, tolerability, pharmacokinetics, and activity of the novel long-acting antimalarial DSM265: a two-part first-in-human phase 1a/1b randomised study. Lancet Infect. Dis. 2017, 17, 626– 635, DOI: 10.1016/S1473-3099(17)30171-8[Crossref], [PubMed], [CAS], Google Scholar427https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXltlWmur8%253D&md5=144cece3cf70a64af921f7f37df7c72dSafety, tolerability, pharmacokinetics, and activity of the novel long-acting antimalarial DSM265: a two-part first-in-human phase 1a/1b randomised studyMcCarthy, James S.; Lotharius, Julie; Ruckle, Thomas; Chalon, Stephan; Phillips, Margaret A.; Elliott, Suzanne; Sekuloski, Silvana; Griffin, Paul; Ng, Caroline L.; Fidock, David A.; Marquart, Louise; Williams, Noelle S.; Gobeau, Nathalie; Bebrevska, Lidiya; Rosario, Maria; Marsh, Kennan; Mohrle, Jorg J.Lancet Infectious Diseases (2017), 17 (6), 626-635CODEN: LIDABP; ISSN:1473-3099. (Elsevier Ltd.)DSM265 is a novel antimalarial that inhibits plasmodial dihydroorotate dehydrogenase, an enzyme essential for pyrimidine biosynthesis. We investigated the safety, tolerability, and pharmacokinetics of DSM265, and tested its antimalarial activity. Healthy participants aged 18-55 years were enrolled in a two-part study: part 1, a single ascending dose (25-1200 mg), double-blind, randomised, placebo-controlled study, and part 2, an open-label, randomised, active-comparator controlled study, in which participants were inoculated with Plasmodium falciparum induced blood-stage malaria (IBSM) and treated with DSM265 (150 mg) or mefloquine (10 mg/kg). Primary endpoints were DSM265 safety, tolerability, and pharmacokinetics. Randomisation lists were created using a validated, automated system. Both parts were registered with the Australian New Zealand Clin. Trials Registry, no. ACTRN12613000522718 (part 1) and no. ACTRN12613000527763 (part 2). In part 1, 73 participants were enrolled between Apr. 12, 2013, and July 14, 2015 (DSM265, n = 55; placebo, n = 18). In part 2, nine participants were enrolled between Sept 30 and Nov 25, 2013 (150 mg DSM265, n = 7; 10 mg/kg mefloquine, n = 2). In part 1, 117 adverse events were reported; no drug-related serious or severe events were reported. The most common drug-related adverse event was headache. The mean DSM265 peak plasma concn. (Cmax) ranged between 1310 ng/mL and 34 800 ng/mL and was reached in a median time (tmax) between 1.5 h and 4 h, with a mean elimination half-life between 86 h and 118 h. In part 2, the log10 parasite redn. ratio at 48 h in the DSM265 (150 mg) group was 1.55 (95% CI 1.42-1.67) and in the mefloquine (10 mg/kg) group was 2.34 (2.17-2.52), corresponding to a parasite clearance half-life of 9.4 h (8.7-10.2) and 6.2 h (5.7-6.7), resp. The median min. inhibitory concn. of DSM265 in blood was estd. as 1040 ng/mL (range 552-1500), resulting in a predicted single efficacious dose of 340 mg. Parasite clearance was significantly faster in participants who received mefloquine than in participants who received DSM265 (p<0.0001). The good safety profile, long elimination half-life, and antimalarial effect of DSM265 supports its development as a partner drug in a single-dose antimalarial combination treatment. Wellcome Trust, UK Department for International Development, Global Health Innovative Technol. Fund, Bill & Melinda Gates Foundation.
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